Through the looking glass: An adventure into the metastable world of the bacterial cytoplasm.

This personal story recounts the accidental observation, the struggles, the breakthroughs, and the collaborative spirit of a few individuals that led to the discovery that bacterial cells expend energy to effectively fluidize their otherwise "glass-like" cytoplasm and promote the dispersal of large cytoplasmic components. This adventure, which led us into an uncharted world at the intersection of cell biology and condensed matter physics about ten years ago, forever transformed the way I view cells and conduct research.

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

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

Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling.

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

Nucleoid Size Scaling and Intracellular Organization of Translation across Bacteria.

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

Subcellular Organization: A Critical Feature of Bacterial Cell Replication.

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

Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

A constant size extension drives bacterial cell size homeostasis.

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

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

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

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

A self-associating protein critical for chromosome attachment, division, and polar organization in caulobacter.

Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB*ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.

Origin of exponential growth in nonlinear reaction networks.

Exponentially growing systems are prevalent in nature, spanning all scales from biochemical reaction networks in single cells to food webs of ecosystems. How exponential growth emerges in nonlinear systems is mathematically unclear. Here, we describe a general theoretical framework that reveals underlying principles of long-term growth: scalability of flux functions and ergodicity of the rescaled systems. Our theory shows that nonlinear fluxes can generate not only balanced growth but also oscillatory or chaotic growth modalities, explaining nonequilibrium dynamics observed in cell cycles and ecosystems. Our mathematical framework is broadly useful in predicting long-term growth rates from natural and synthetic networks, analyzing the effects of system noise and perturbations, validating empirical and phenomenological laws on growth rate, and studying autocatalysis and network evolution.

A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis.

Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNγ, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host's response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts.

Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

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

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

A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen.IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of α-ketoglutarate.

To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

Pathogen-mediated manipulation of arthropod microbiota to promote infection.

Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli.

Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

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

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