Self-enhanced mobility enables vortex pattern formation in living matter.

Ranging from subcellular organelle biogenesis to embryo development, the formation of self-organized structures is a hallmark of living systems. Whereas the emergence of ordered spatial patterns in biology is often driven by intricate chemical signalling that coordinates cellular behaviour and differentiation1-4, purely physical interactions can drive the formation of regular biological patterns such as crystalline vortex arrays in suspensions of spermatozoa5 and bacteria6. Here we discovered a new route to self-organized pattern formation driven by physical interactions, which creates large-scale regular spatial structures with multiscale ordering. Specifically we found that dense bacterial living matter spontaneously developed a lattice of mesoscale, fast-spinning vortices; these vortices each consisted of around 104-105 motile bacterial cells and were arranged in space at greater than centimetre scale and with apparent hexagonal order, whereas individual cells in the vortices moved in coordinated directions with strong polar and vortical order. Single-cell tracking and numerical simulations suggest that the phenomenon is enabled by self-enhanced mobility in the system-that is, the speed of individual cells increasing with cell-generated collective stresses at a given cell density. Stress-induced mobility enhancement and fluidization is prevalent in dense living matter at various scales of length7-9. Our findings demonstrate that self-enhanced mobility offers a simple physical mechanism for pattern formation in living systems and, more generally, in other active matter systems10 near the boundary of fluid- and solid-like behaviours11-17.

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.

Learning from the unknown: exploring the range of bacterial functionality.

Determining the repertoire of a microbe's molecular functions is a central question in microbial biology. Modern techniques achieve this goal by comparing microbial genetic material against reference databases of functionally annotated genes/proteins or known taxonomic markers such as 16S rRNA. Here, we describe a novel approach to exploring bacterial functional repertoires without reference databases. Our Fusion scheme establishes functional relationships between bacteria and assigns organisms to Fusion-taxa that differ from otherwise defined taxonomic clades. Three key findings of our work stand out. First, bacterial functional comparisons outperform marker genes in assigning taxonomic clades. Fusion profiles are also better for this task than other functional annotation schemes. Second, Fusion-taxa are robust to addition of novel organisms and are, arguably, able to capture the environment-driven bacterial diversity. Finally, our alignment-free nucleic acid-based Siamese Neural Network model, created using Fusion functions, enables finding shared functionality of very distant, possibly structurally different, microbial homologs. Our work can thus help annotate functional repertoires of bacterial organisms and further guide our understanding of microbial communities.

Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

Hydration solids.

Hygroscopic biological matter in plants, fungi and bacteria make up a large fraction of Earth's biomass1. Although metabolically inert, these water-responsive materials exchange water with the environment and actuate movement2-5 and have inspired technological uses6,7. Despite the variety in chemical composition, hygroscopic biological materials across multiple kingdoms of life exhibit similar mechanical behaviours including changes in size and stiffness with relative humidity8-13. Here we report atomic force microscopy measurements on the hygroscopic spores14,15 of a common soil bacterium and develop a theory that captures the observed equilibrium, non-equilibrium and water-responsive mechanical behaviours, finding that these are controlled by the hydration force16-18. Our theory based on the hydration force explains an extreme slowdown of water transport and successfully predicts a strong nonlinear elasticity and a transition in mechanical properties that differs from glassy and poroelastic behaviours. These results indicate that water not only endows biological matter with fluidity but also can-through the hydration force-control macroscopic properties and give rise to a 'hydration solid' with unusual properties. A large fraction of biological matter could belong to this distinct class of solid matter.

Stochastic Monte Carlo Model for Simulating the Dynamic Liquid-Liquid Phase Separation in Bacterial Cells.

There is growing evidence showing that many critical biological processes are driven by biomolecule condensates through liquid-liquid phase separation (LLPS). Although the qualitative observation and description of LLPS have been well documented, quantitative simulations of the time-dependent progression of LLPS in live cells are generally lacking. In this work, we build a stochastic Monte Carlo model to simulate the dynamic LLPS process during the formation of bacterial aggresomes. We demonstrate that the size distribution of the protein condensates evolves from an exponential-like to a bimodal-like pattern, and the number of condensates increases at the beginning and then decreases after reaching a maximum. Incorporating diffusion and collision, our simplified model recapitulates the two-step LLPS process in which many smaller condensates are formed in the first step and then merged into a few larger ones. We further reveal that the condensation speed, which can be defined by the condensates formed in unit time during the first step, is mainly determined by both the collision energy barrier and the initial protein density, while the number of condensates at the equilibrium is mainly associated with the dissociation energy barrier. Moreover, the LLPS process is not sensitive to temperature changes ranging around physiological conditions. Additionally, we consider the effect of the nucleation energy barrier on LLPS. We find that a higher nucleation energy barrier brings a slower condensation speed. Overall, we simulate the spatiotemporal dynamics of the LLPS process and provide qualitative guidance for understanding the dynamics of LLPS in bacterial cells, which can faithfully recapitulate experimental observations and facilitate the design of future experimental tests.

Pressure-induced shape-shifting of helical bacteria.

Many bacterial species are helical in shape, including the widespread pathogen H. pylori. Motivated by recent experiments on H. pylori showing that cell wall synthesis is not uniform [J. A. Taylor, et al., eLife, 2020, 9, e52482], we investigate the possible formation of helical cell shape induced by elastic heterogeneity. We show, experimentally and theoretically, that helical morphogenesis can be produced by pressurizing an elastic cylindrical vessel with helical reinforced lines. The properties of the pressurized helix are highly dependent on the initial helical angle of the reinforced region. We find that steep angles result in crooked helices with, surprisingly, a reduced end-to-end distance upon pressurization. This work helps explain the possible mechanisms for the generation of helical cell morphologies and may inspire the design of novel pressure-controlled helical actuators.

Interaction of the bacterial division regulator MinE with lipid bicelles studied by NMR spectroscopy.

The bacterial MinE and MinD division regulatory proteins form a standing wave enabling MinC, which binds MinD, to inhibit FtsZ polymerization everywhere except at the midcell, thereby assuring correct positioning of the cytokinetic septum and even distribution of contents to daughter cells. The MinE dimer undergoes major structural rearrangements between a resting six-stranded state present in the cytoplasm, a membrane-bound state, and a four-stranded active state bound to MinD on the membrane, but it is unclear which MinE motifs interact with the membrane in these different states. Using NMR, we probe the structure and global dynamics of MinE bound to disc-shaped lipid bicelles. In the bicelle-bound state, helix α1 no longer sits on top of the six-stranded ß-sheet, losing any contact with the protein core, but interacts directly with the bicelle surface; the structure of the protein core remains unperturbed and also interacts with the bicelle surface via helix α2. Binding may involve a previously identified excited state of free MinE in which helix α1 is disordered, thereby allowing it to target the membrane surface. Helix α1 and the protein core undergo nanosecond rigid body motions of differing amplitudes in the plane of the bicelle surface. Global dynamics on the sub-millisecond time scale between a ground state and a sparsely populated excited state are also observed and may represent a very early intermediate on the transition path between the resting six-stranded and active four-stranded conformations. In summary, our results provide insights into MinE structural rearrangements important during bacterial cell division.

Antibacterial Activity and Mechanism of Oxidized Bacterial Nanocellulose with Different Carboxyl Content.

Oxidized bacterial nanocellulose (OBC) is reported to prevent microbial growth, but its antibacterial characteristics and mechanism are still unclear. Here, the antibacterial mechanism of OBC is explored by detecting and assessing the interaction of OBC with different carboxyl content on Staphylococcus aureus and Escherichia coli. The results show that OBC has strong antibacterial activity and antibiofilm activity against S. aureus and E. coli, which is positively correlated with the carboxyl content of OBC. After OBC treatment, the bacteria adhesion is inhibited and the cell membrane is destroyed leading to increased permeability. Further investigation reveals that the concentration of cyclic diguanosine monophosphate (c-di-GMP) that induced biofilm formation is significantly decreased to 1.81 pmol mg-1 after OBC treatment. In addition, OBC inactivates mature biofilms, with inactivation rates up to 79.3%. This study suggests that OBC has excellent antibacterial and antiadhesion properties, which can increase the cell membrane permeability and inhibit c-di-GMP formation. In addition, OBC also has a strong inactivation effect on mature biofilm, which can be used as an effective antibiofilm agent.

Architecture and self-assembly of the jumbo bacteriophage nuclear shell.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Structure and Mechanism of the Lipid Flippase MurJ.

Biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates the transport of lipid-linked oligosaccharides (LLO) across membranes from their cytosolic site of synthesis to their sites of utilization. Much of our current understanding of LLO transport comes from genetic, biochemical, and structural studies of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily protein MurJ, which flips the peptidoglycan precursor lipid II. MurJ plays a pivotal role in bacterial cell wall synthesis and is an emerging antibiotic target. Here, we review the mechanism of LLO flipping by MurJ, including the structural basis for lipid II flipping and ion coupling. We then discuss inhibition of MurJ by antibacterials, including humimycins and the phage M lysis protein, as well as how studies on MurJ could provide insight into other flippases, both within and beyond the MOP superfamily.

Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.

Connectivity of Fennoscandian Shield terrestrial deep biosphere microbiomes with surface communities.

The deep biosphere is an energy constrained ecosystem yet fosters diverse microbial communities that are key in biogeochemical cycling. Whether microbial communities in deep biosphere groundwaters are shaped by infiltration of allochthonous surface microorganisms or the evolution of autochthonous species remains unresolved. In this study, 16S rRNA gene amplicon analyses showed that few groups of surface microbes infiltrated deep biosphere groundwaters at the Äspö Hard Rock Laboratory, Sweden, but that such populations constituted up to 49% of the microbial abundance. The dominant persisting phyla included Patescibacteria, Proteobacteria, and Epsilonbacteraeota. Despite the hydrological connection of the Baltic Sea with the studied groundwaters, infiltrating microbes predominantly originated from deep soil groundwater. Most deep biosphere groundwater populations lacked surface representatives, suggesting that they have evolved from ancient autochthonous populations. We propose that deep biosphere groundwater communities in the Fennoscandian Shield consist of selected infiltrated and indigenous populations adapted to the prevailing conditions.

DeLTA 2.0: A deep learning pipeline for quantifying single-cell spatial and temporal dynamics.

Improvements in microscopy software and hardware have dramatically increased the pace of image acquisition, making analysis a major bottleneck in generating quantitative, single-cell data. Although tools for segmenting and tracking bacteria within time-lapse images exist, most require human input, are specialized to the experimental set up, or lack accuracy. Here, we introduce DeLTA 2.0, a purely Python workflow that can rapidly and accurately analyze images of single cells on two-dimensional surfaces to quantify gene expression and cell growth. The algorithm uses deep convolutional neural networks to extract single-cell information from time-lapse images, requiring no human input after training. DeLTA 2.0 retains all the functionality of the original version, which was optimized for bacteria growing in the mother machine microfluidic device, but extends results to two-dimensional growth environments. Two-dimensional environments represent an important class of data because they are more straightforward to implement experimentally, they offer the potential for studies using co-cultures of cells, and they can be used to quantify spatial effects and multi-generational phenomena. However, segmentation and tracking are significantly more challenging tasks in two-dimensions due to exponential increases in the number of cells. To showcase this new functionality, we analyze mixed populations of antibiotic resistant and susceptible cells, and also track pole age and growth rate across generations. In addition to the two-dimensional capabilities, we also introduce several major improvements to the code that increase accessibility, including the ability to accept many standard microscopy file formats as inputs and the introduction of a Google Colab notebook so users can try the software without installing the code on their local machine. DeLTA 2.0 is rapid, with run times of less than 10 minutes for complete movies with hundreds of cells, and is highly accurate, with error rates around 1%, making it a powerful tool for analyzing time-lapse microscopy data.

Bacterial Small Membrane Proteins: the Swiss Army Knife of Regulators at the Lipid Bilayer.

Small membrane proteins represent a subset of recently discovered small proteins (≤100 amino acids), which are a ubiquitous class of emerging regulators underlying bacterial adaptation to environmental stressors. Until relatively recently, small open reading frames encoding these proteins were not designated genes in genome annotations. Therefore, our understanding of small protein biology was primarily limited to a few candidates associated with previously characterized larger partner proteins. Following the first systematic analyses of small proteins in Escherichia coli over a decade ago, numerous small proteins across different bacteria have been uncovered. An estimated one-third of these newly discovered proteins in E. coli are localized to the cell membrane, where they may interact with distinct groups of membrane proteins, such as signal receptors, transporters, and enzymes, and affect their activities. Recently, there has been considerable progress in functionally characterizing small membrane protein regulators aided by innovative tools adapted specifically to study small proteins. Our review covers prototypical proteins that modulate a broad range of cellular processes, such as transport, signal transduction, stress response, respiration, cell division, sporulation, and membrane stability. Thus, small membrane proteins represent a versatile group of physiology regulators at the membrane and the whole cell. Additionally, small membrane proteins have the potential for clinical applications, where some of the proteins may act as antibacterial agents themselves while others serve as alternative drug targets for the development of novel antimicrobials.

Tailoring Surface Properties of Electrodes for Synchronous Enhanced Extracellular Electron Transfer and Enriched Exoelectrogens in Microbial Fuel Cells.

An extracellular electron transfer (EET) process between an electroactive biofilm and an electrode is a crucial step for the performance of microbial fuel cells (MFCs), which is highly related to the enrichment of exoelectrogens and the electrocatalytic activity of the electrode. Herein, an efficient N- and Fe-abundant carbon cloth (CC) electrode with the comodification of iron porphyrin (FePor) and polyquaternium-7 (PQ) was synthesized using a facile solvent evaporation and immersion method and developed as an anode (named FePor-PQ) in MFCs. The surface structural characterizations confirmed the successful introduction of N and Fe atoms, whereas FePor-PQ achieved the N content of 9.59 at %, which may offer various active sites for EET. The introduction of PQ contributed to improving the surface hydrophilicity, providing the composite electrode good biocompatibility for bacterial attachment and colonization as well as substrate diffusion. Based on the advantages, the MFC with the FePor-PQ anode produced a maximum power density of 2165.7 mW m-2, strikingly higher than those of CC (1124.0 mW m-2), PQ (1668.8 mW m-2), and FePor (1978.9 mW m-2). Furthermore, with the EET mediated by the binding of flavins and c-type cytochromes on the outer membrane was enhanced prominently, the typical exoelectrogen Geobacter was enriched up to 55.84% in the FePor-PQ anode biofilm. This work reveals a synergistic effect from heteroatom coating and surface properties tailoring to boost both the EET efficiency and exoelectrogen enrichment for enhancing MFC performance, which also provides valuable insights for designing electrodes in other bio-electrochemical systems.

Dense intracellular ion pools in unicellular organisms.

Uptake and concentration of inorganic ions are part of the complex cellular processes required for cell homeostasis, as well as for mineral formation by organisms. These ion transport mechanisms include distinct cellular compartments and chemical phases that play various roles in the physiology of organisms. Here, the prominent cases of dense ion pools in unicellular organisms are briefly reviewed. The specific observations that were reported for different organisms are consolidated into a wide perspective that emphasizes general traits. It is suggested that the intracellular ion pools can be divided into three types: a high cytoplasmic concentration, a labile storage compartment that hosts dense ion-rich phases, and a mineral-forming compartment in which a stable long-lived structure is formed. Recently, many labile pools were identified in various organisms using advanced techniques, bringing many new questions about their possible roles in the formation of the stable mineralized structures.