Capillary interactions drive the self-organization of bacterial colonies.

Many bacteria inhabit thin layers of water on solid surfaces both naturally in soils or on hosts or textiles and in the lab on agar hydrogels. In these environments, cells experience capillary forces, yet an understanding of how these forces shape bacterial collective behaviors remains elusive. Here, we show that the water menisci formed around bacteria lead to capillary attraction between cells while still allowing them to slide past one another. We develop an experimental apparatus that allows us to control bacterial collective behaviors by varying the strength and range of capillary forces. Combining 3D imaging and cell tracking with agent-based modeling, we demonstrate that capillary attraction organizes rod-shaped bacteria into densely packed, nematic groups, and profoundly influences their collective dynamics and morphologies. Our results suggest that capillary forces may be a ubiquitous physical ingredient in shaping microbial communities in partially hydrated environments.

Temperature compensation through kinetic regulation in biochemical oscillators.

Nearly all circadian clocks maintain a period that is insensitive to temperature changes, a phenomenon known as temperature compensation (TC). Yet, it is unclear whether there is any common feature among different systems that exhibit TC. From a general timescale invariance, we show that TC relies on the existence of certain period-lengthening reactions wherein the period of the system increases strongly with the rates in these reactions. By studying several generic oscillator models, we show that this counterintuitive dependence is nonetheless a common feature of oscillators in the nonlinear (far-from-onset) regime where the oscillation can be separated into fast and slow phases. The increase of the period with the period-lengthening reaction rates occurs when the amplitude of the slow phase in the oscillation increases with these rates while the progression speed in the slow phase is controlled by other rates of the system. The positive dependence of the period on the period-lengthening rates balances its inverse dependence on other kinetic rates in the system, which gives rise to robust TC in a wide range of parameters. We demonstrate the existence of such period-lengthening reactions and their relevance for TC in all four model systems we considered. Theoretical results for a model of the Kai system are supported by experimental data. A study of the energy dissipation also shows that better TC performance requires higher energy consumption. Our study unveils a general mechanism by which a biochemical oscillator achieves TC by operating in parameter regimes far from the onset where period-lengthening reactions exist.

Mechanical constraints to unbound expansion of B. subtilis on semi-solid surfaces.

IMPORTANCE: How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

Local polar order controls mechanical stress and triggers layer formation in developing Myxococcus xanthus colonies.

Colonies of the social bacterium Myxococcus xanthus go through a morphological transition from a thin colony of cells to three-dimensional droplet-like fruiting bodies as a strategy to survive starvation. The biological pathways that control the decision to form a fruiting body have been studied extensively. However, the mechanical events that trigger the creation of multiple cell layers and give rise to droplet formation remain poorly understood. By measuring cell orientation, velocity, polarity, and force with cell-scale resolution, we reveal a stochastic local polar order in addition to the more obvious nematic order. Average cell velocity and active force at topological defects agree with predictions from active nematic theory, but their fluctuations are anomalously large due to polar active forces generated by the self-propelled rod-shaped cells. We find that M. xanthus cells adjust their reversal frequency to tune the magnitude of this local polar order, which in turn controls the mechanical stresses and triggers layer formation in the colonies.

Natural silencing of quorum-sensing activity protects Vibrio parahaemolyticus from lysis by an autoinducer-detecting phage.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.

Natural Silencing of Quorum-Sensing Activity Protects Vibrio parahaemolyticus from Lysis by an Autoinducer-Detecting Phage.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip . Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR . VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO , encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.

Subcellular localization of type IV pili regulates bacterial multicellular development.

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities.

New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields.

Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.

Modelling the pyrenoid-based CO2-concentrating mechanism provides insights into its operating principles and a roadmap for its engineering into crops.

Many eukaryotic photosynthetic organisms enhance their carbon uptake by supplying concentrated CO2 to the CO2-fixing enzyme Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer this pyrenoid-based CO2-concentrating mechanism (PCCM) into crops to increase yields. Here we develop a computational model for a PCCM on the basis of the postulated mechanism in the green alga Chlamydomonas reinhardtii. Our model recapitulates all Chlamydomonas PCCM-deficient mutant phenotypes and yields general biophysical principles underlying the PCCM. We show that an effective and energetically efficient PCCM requires a physical barrier to reduce pyrenoid CO2 leakage, as well as proper enzyme localization to reduce futile cycling between CO2 and HCO3-. Importantly, our model demonstrates the feasibility of a purely passive CO2 uptake strategy at air-level CO2, while active HCO3- uptake proves advantageous at lower CO2 levels. We propose a four-step engineering path to increase the rate of CO2 fixation in the plant chloroplast up to threefold at a theoretical cost of only 1.3 ATP per CO2 fixed, thereby offering a framework to guide the engineering of a PCCM into land plants.

Quantitative input-output dynamics of a c-di-GMP signal transduction cascade in Vibrio cholerae.

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

LuxT Is a Global Regulator of Low-Cell-Density Behaviors, Including Type III Secretion, Siderophore Production, and Aerolysin Production, in Vibrio harveyi.

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and posttranscriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the TetR family transcriptional regulator LuxT was shown to control Vibrio harveyi qrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA sequencing to reveal that, beyond the control of qrr1, LuxT is a global regulator of 414 V. harveyi genes, including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a GntR family transcriptional regulator, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ dependent and one that is SwrZ independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low-cell-density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case, we find that in Aliivibrio fischeri, LuxT also represses swrZ. SwrZ is a repressor of A. fischeri siderophore production genes. Thus, LuxT repression of swrZ drives the activation of A. fischeri siderophore gene expression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression. IMPORTANCE Bacteria precisely tune gene expression patterns to successfully react to changes that occur in the environment. Defining the mechanisms that enable bacteria to thrive in diverse and fluctuating habitats, including in host organisms, is crucial for a deep understanding of the microbial world and also for the development of effective applications to promote or combat particular bacteria. In this study, we show that a regulator called LuxT controls over 400 genes in the marine bacterium Vibrio harveyi and that LuxT is highly conserved among Vibrionaceae species, ubiquitous marine bacteria that often cause disease. We characterize the mechanisms by which LuxT controls genes involved in virulence and nutrient acquisition. We show that LuxT functions in parallel with a set of regulators of the bacterial cell-to-cell communication process called quorum sensing to promote V. harveyi behaviors at low cell density.

Hierarchical transitions and fractal wrinkling drive bacterial pellicle morphogenesis.

Bacterial cells can self-organize into structured communities at fluid-fluid interfaces. These soft, living materials composed of cells and extracellular matrix are called pellicles. Cells residing in pellicles garner group-level survival advantages such as increased antibiotic resistance. The dynamics of pellicle formation and, more generally, how complex morphologies arise from active biomaterials confined at interfaces are not well understood. Here, using Vibrio cholerae as our model organism, a custom-built adaptive stereo microscope, fluorescence imaging, mechanical theory, and simulations, we report a fractal wrinkling morphogenesis program that differs radically from the well-known coalescence of wrinkles into folds that occurs in passive thin films at fluid-fluid interfaces. Four stages occur: growth of founding colonies, onset of primary wrinkles, development of secondary curved ridge instabilities, and finally the emergence of a cascade of finer structures with fractal-like scaling in wavelength. The time evolution of pellicle formation depends on the initial heterogeneity of the film microstructure. Changing the starting bacterial seeding density produces three variations in the sequence of morphogenic stages, which we term the bypass, crystalline, and incomplete modes. Despite these global architectural transitions, individual microcolonies remain spatially segregated, and thus, the community maintains spatial and genetic heterogeneity. Our results suggest that the memory of the original microstructure is critical in setting the morphogenic dynamics of a pellicle as an active biomaterial.

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA.

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili.

Type IV pili (TFP) function through cycles of extension and retraction. The coordination of these cycles remains mysterious due to a lack of quantitative measurements of multiple features of TFP dynamics. Here, we fluorescently label TFP in the pathogen Pseudomonas aeruginosa and track full extension and retraction cycles of individual filaments. Polymerization and depolymerization dynamics are stochastic; TFP are made at random times and extend, pause, and retract for random lengths of time. TFP can also pause for extended periods between two extension or two retraction events in both wild-type cells and a slowly retracting PilT mutant. We developed a biophysical model based on the stochastic binding of two dedicated extension and retraction motors to the same pilus machine that predicts the observed features of the data with no free parameters. We show that only a model in which both motors stochastically bind and unbind to the pilus machine independent of the piliation state of the machine quantitatively explains the experimentally observed pilus production rate. In experimental support of this model, we show that the abundance of the retraction motor dictates the pilus production rate and that PilT is bound to pilus machines even in their unpiliated state. Together, the strong quantitative agreement of our model with a variety of experiments suggests that the entire repetitive cycle of pilus extension and retraction is coordinated by the competition of stochastic motor binding to the pilus machine, and that the retraction motor is the major throttle for pilus production.

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal.

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulating V. cholerae biofilm dispersal to ameliorate disease.

Cell position fates and collective fountain flow in bacterial biofilms revealed by light-sheet microscopy.

Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell.

Nonuniform growth and surface friction determine bacterial biofilm morphology on soft substrates.

During development, organisms acquire three-dimensional (3D) shapes with important physiological consequences. While basic mechanisms underlying morphogenesis are known in eukaryotes, it is often difficult to manipulate them in vivo. To circumvent this issue, here we present a study of developing Vibrio cholerae biofilms grown on agar substrates in which the spatiotemporal morphological patterns were altered by varying the agar concentration. Expanding biofilms are initially flat but later undergo a mechanical instability and become wrinkled. To gain mechanistic insights into this dynamic pattern-formation process, we developed a model that considers diffusion of nutrients and their uptake by bacteria, bacterial growth/biofilm matrix production, mechanical deformation of both the biofilm and the substrate, and the friction between them. Our model shows quantitative agreement with experimental measurements of biofilm expansion dynamics, and it accurately predicts two distinct spatiotemporal patterns observed in the experiments-the wrinkles initially appear either in the peripheral region and propagate inward (soft substrate/low friction) or in the central region and propagate outward (stiff substrate/high friction). Our results, which establish that nonuniform growth and friction are fundamental determinants of stress anisotropy and hence biofilm morphology, are broadly applicable to bacterial biofilms with similar morphologies and also provide insight into how other bacterial biofilms form distinct wrinkle patterns. We discuss the implications of forming undulated biofilm morphologies, which may enhance the availability of nutrients and signaling molecules and serve as a "bet hedging" strategy.

Mechanical instability and interfacial energy drive biofilm morphogenesis.

Surface-attached bacterial communities called biofilms display a diversity of morphologies. Although structural and regulatory components required for biofilm formation are known, it is not understood how these essential constituents promote biofilm surface morphology. Here, using Vibrio cholerae as our model system, we combine mechanical measurements, theory and simulation, quantitative image analyses, surface energy characterizations, and mutagenesis to show that mechanical instabilities, including wrinkling and delamination, underlie the morphogenesis program of growing biofilms. We also identify interfacial energy as a key driving force for mechanomorphogenesis because it dictates the generation of new and the annihilation of existing interfaces. Finally, we discover feedback between mechanomorphogenesis and biofilm expansion, which shapes the overall biofilm contour. The morphogenesis principles that we discover in bacterial biofilms, which rely on mechanical instabilities and interfacial energies, should be generally applicable to morphogenesis processes in tissues in higher organisms.