Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Escherichia coli in Auto(trophic) Mode.

It is challenging to convert a heterotrophic organism that loves sugars and other multicarbon compounds as energy and carbon sources into an autotroph that builds all biomass from carbon dioxide. In this issue, Gleizer et al. demonstrate how this can be achieved.

Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection.

The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here, we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transitions between subpopulations and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers and therapeutic targets at single-cell resolution.

The Synchronization of Replication and Division Cycles in Individual E. coli Cells.

Isogenic E. coli cells growing in a constant environment display significant variability in growth rates, division sizes, and generation times. The guiding principle appears to be that each cell, during one generation, adds a size increment that is uncorrelated to its birth size. Here, we investigate the mechanisms underlying this "adder" behavior by mapping the chromosome replication cycle to the division cycle of individual cells using fluorescence microscopy. We have found that initiation of chromosome replication is triggered at a fixed volume per chromosome independent of a cell's birth volume and growth rate. Each initiation event is coupled to a division event after a growth-rate-dependent time. We formalize our findings in a model showing that cell-to-cell variation in division timing and cell size is mainly driven by variations in growth rate. The model also explains why fast-growing cells display adder behavior and correctly predict deviations from the adder behavior at slow growth.

Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings.

Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex.

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F "sex" pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.

The mechanical world of bacteria.

In the wild, bacteria are predominantly associated with surfaces as opposed to existing as free-swimming, isolated organisms. They are thus subject to surface-specific mechanics, including hydrodynamic forces, adhesive forces, the rheology of their surroundings, and transport rules that define their encounters with nutrients and signaling molecules. Here, we highlight the effects of mechanics on bacterial behaviors on surfaces at multiple length scales, from single bacteria to the development of multicellular bacterial communities such as biofilms.

An integrated approach reveals regulatory controls on bacterial translation elongation.

Ribosomes elongate at a nonuniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally constrained framework for modeling translation.

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.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

Four-dimensional imaging of E. coli nucleoid organization and dynamics in living cells.

Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (1) Nucleoid density coalesces into longitudinal bundles, giving a stiff, low-DNA-density ellipsoid. (2) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and directs global nucleoid dynamics, including sister segregation. (3) Longitudinal density waves flux back and forth along the nucleoid, with 5%-10% of density shifting within 5 s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5%-15%. Pulses occur at 20 min intervals, at defined cell-cycle times. This progression includes sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intranucleoid mechanical stress. These effects could comprise a chromosome-based cell-cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

Contingency and statistical laws in replicate microbial closed ecosystems.

Contingency, the persistent influence of past random events, pervades biology. To what extent, then, is each course of ecological or evolutionary dynamics unique, and to what extent are these dynamics subject to a common statistical structure? Addressing this question requires replicate measurements to search for emergent statistical laws. We establish a readily replicated microbial closed ecosystem (CES), sustaining its three species for years. We precisely measure the local population density of each species in many CES replicates, started from the same initial conditions and kept under constant light and temperature. The covariation among replicates of the three species densities acquires a stable structure, which could be decomposed into discrete eigenvectors, or "ecomodes." The largest ecomode dominates population density fluctuations around the replicate-average dynamics. These fluctuations follow simple power laws consistent with a geometric random walk. Thus, variability in ecological dynamics can be studied with CES replicates and described by simple statistical laws.

A self-produced trigger for biofilm disassembly that targets exopolysaccharide.

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli, and Staphylococcus aureus.

Viscoelastic control of spatiotemporal order in bacterial active matter.

Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.

Mechanics limits ecological diversity and promotes heterogeneity in confined bacterial communities.

Multispecies bacterial populations often inhabit confined and densely packed environments where spatial competition determines the ecological diversity of the community. However, the role of mechanical interactions in shaping the ecology is still poorly understood. Here, we study a model system consisting of two populations of nonmotile Escherichia coli bacteria competing within open, monolayer microchannels. The competitive dynamics is observed to be biphasic: After seeding, either one strain rapidly fixates or both strains orient into spatially stratified, stable communities. We find that mechanical interactions with other cells and local spatial constraints influence the resulting community ecology in unexpected ways, severely limiting the overall diversity of the communities while simultaneously allowing for the establishment of stable, heterogeneous populations of bacteria displaying disparate growth rates. Surprisingly, the populations have a high probability of coexisting even when one strain has a significant growth advantage. A more coccus morphology is shown to provide a selective advantage, but agent-based simulations indicate this is due to hydrodynamic and adhesion effects within the microchannel and not from breaking of the nematic ordering. Our observations are qualitatively reproduced by a simple Pólya urn model, which suggests the generality of our findings for confined population dynamics and highlights the importance of early colonization conditions on the resulting diversity and ecology of bacterial communities. These results provide fundamental insights into the determinants of community diversity in dense confined ecosystems where spatial exclusion is central to competition as in organized biofilms or intestinal crypts.

My life with nature.

After a childhood in Germany and being a youth in Grand Forks, North Dakota, I went to Harvard University, then to graduate school in biochemistry at the University of Wisconsin. Then to Washington University and Stanford University for postdoctoral training in biochemistry and genetics. Then at the University of Wisconsin, as a professor in the Department of Biochemistry and the Department of Genetics, I initiated research on bacterial chemotaxis. Here, I review this research by me and by many, many others up to the present moment. During the past few years, I have been studying chemotaxis and related behavior in animals, namely in Drosophila fruit flies, and some of these results are presented here. My current thinking is described.

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Thermal robustness of signaling in bacterial chemotaxis.

Temperature is a global factor that affects the performance of all intracellular networks. Robustness against temperature variations is thus expected to be an essential network property, particularly in organisms without inherent temperature control. Here, we combine experimental analyses with computational modeling to investigate thermal robustness of signaling in chemotaxis of Escherichia coli, a relatively simple and well-established model for systems biology. We show that steady-state and kinetic pathway parameters that are essential for chemotactic performance are indeed temperature-compensated in the entire physiological range. Thermal robustness of steady-state pathway output is ensured at several levels by mutual compensation of temperature effects on activities of individual pathway components. Moreover, the effect of temperature on adaptation kinetics is counterbalanced by preprogrammed temperature dependence of enzyme synthesis and stability to achieve nearly optimal performance at the growth temperature. Similar compensatory mechanisms are expected to ensure thermal robustness in other systems.

A primary role for release factor 3 in quality control during translation elongation in Escherichia coli.

Release factor 3 (RF3) is a GTPase found in a broad range of bacteria where it is thought to play a critical "recycling" role in translation by facilitating the removal of class 1 release factors (RF1 and RF2) from the ribosome following peptide release. More recently, RF3 was shown in vitro to stimulate a retrospective editing reaction on the bacterial ribosome wherein peptides carrying mistakes are prematurely terminated during protein synthesis. Here, we examine the role of RF3 in the bacterial cell and show that the deletion of this gene sensitizes cells to other perturbations that reduce the overall fidelity of protein synthesis. We further document substantial effects on mRNA stability and protein expression using reporter systems, native mRNAs and proteins. We conclude that RF3 plays a primary role in vivo in specifying the fidelity of protein synthesis thus impacting overall protein quantity and quality.

Bacterial coexistence driven by motility and spatial competition.

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology1,2 and microbiome research3. Bacteria are known to coexist by metabolic specialization4, cooperation5 and cyclic warfare6-8. Many species are also motile9, which is studied in terms of mechanism10,11, benefit12,13, strategy14,15, evolution16,17 and ecology18,19. Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances2,20,21. However, the role of bacterial motility in coexistence remains unexplored experimentally. Here we show that-for mixed bacterial populations that colonize nutrient patches-either population outcompetes the other when low in relative abundance. This inversion of the competitive hierarchy is caused by active segregation and spatial exclusion within the patch: a small fast-moving population can outcompete a large fast-growing population by impeding its migration into the patch, while a small fast-growing population can outcompete a large fast-moving population by expelling it from the initial contact area. The resulting spatial segregation is lost for weak growth-migration trade-offs and a lack of virgin space, but is robust to population ratio, density and chemotactic ability, and is observed in both laboratory and wild strains. These findings show that motility differences and their trade-offs with growth are sufficient to promote diversity, and suggest previously undescribed roles for motility in niche formation and collective expulsion-containment strategies beyond individual search and survival.