Pleiotropic hubs drive bacterial surface competition through parallel changes in colony composition and expansion.

Bacteria commonly adhere to surfaces where they compete for both space and resources. Despite the importance of surface growth, it remains largely elusive how bacteria evolve on surfaces. We previously performed an evolution experiment where we evolved distinct Bacilli populations under a selective regime that favored colony spreading. In just a few weeks, colonies of Bacillus subtilis showed strongly advanced expansion rates, increasing their radius 2.5-fold relative to that of the ancestor. Here, we investigate what drives their rapid evolution by performing a uniquely detailed analysis of the evolutionary changes in colony development. We find mutations in diverse global regulators, RicT, RNAse Y, and LexA, with strikingly similar pleiotropic effects: They lower the rate of sporulation and simultaneously facilitate colony expansion by either reducing extracellular polysaccharide production or by promoting filamentous growth. Combining both high-throughput flow cytometry and gene expression profiling, we show that regulatory mutations lead to highly reproducible and parallel changes in global gene expression, affecting approximately 45% of all genes. This parallelism results from the coordinated manner by which regulators change activity both during colony development-in the transition from vegetative growth to dormancy-and over evolutionary time. This coordinated activity can however also break down, leading to evolutionary divergence. Altogether, we show how global regulators function as major pleiotropic hubs that drive rapid surface adaptation by mediating parallel changes in both colony composition and expansion, thereby massively reshaping gene expression.

Lineage Tracing and Single-Nucleus Multiomics Reveal Novel Features of Adaptive and Maladaptive Repair after Acute Kidney Injury.

SIGNIFICANCE STATEMENT: Understanding the mechanisms underlying adaptive and maladaptive renal repair after AKI and their long-term consequences is critical to kidney health. The authors used lineage tracing of cycling cells and single-nucleus multiomics (profiling transcriptome and chromatin accessibility) after AKI. They demonstrated that AKI triggers a cell-cycle response in most epithelial and nonepithelial kidney cell types. They also showed that maladaptive proinflammatory proximal tubule cells (PTCs) persist until 6 months post-AKI, although they decreased in abundance over time, in part, through cell death. Single-nucleus multiomics of lineage-traced cells revealed regulatory features of adaptive and maladaptive repair. These included activation of cell state-specific transcription factors and cis-regulatory elements, and effects in PTCs even after adaptive repair, weeks after the injury event. BACKGROUND: AKI triggers a proliferative response as part of an intrinsic cellular repair program, which can lead to adaptive renal repair, restoring kidney structure and function, or maladaptive repair with the persistence of injured proximal tubule cells (PTCs) and an altered kidney structure. However, the cellular and molecular understanding of these repair programs is limited. METHODS: To examine chromatin and transcriptional responses in the same cell upon ischemia-reperfusion injury (IRI), we combined genetic fate mapping of cycling ( Ki67+ ) cells labeled early after IRI with single-nucleus multiomics-profiling transcriptome and chromatin accessibility in the same nucleus-and generated a dataset of 83,315 nuclei. RESULTS: AKI triggered a broad cell cycle response preceded by cell type-specific and global transcriptional changes in the nephron, the collecting and vascular systems, and stromal and immune cell types. We observed a heterogeneous population of maladaptive PTCs throughout proximal tubule segments 6 months post-AKI, with a marked loss of maladaptive cells from 4 weeks to 6 months. Gene expression and chromatin accessibility profiling in the same nuclei highlighted differences between adaptive and maladaptive PTCs in the activity of cis-regulatory elements and transcription factors, accompanied by corresponding changes in target gene expression. Adaptive repair was associated with reduced expression of genes encoding transmembrane transport proteins essential to kidney function. CONCLUSIONS: Analysis of genome organization and gene activity with single-cell resolution using lineage tracing and single-nucleus multiomics offers new insight into the regulation of renal injury repair. Weeks to months after mild-to-moderate IRI, maladaptive PTCs persist with an aberrant epigenetic landscape, and PTCs exhibit an altered transcriptional profile even following adaptive repair.

Complex extracellular biology drives surface competition during colony expansion in Bacillus subtilis.

Many bacteria grow on surfaces in nature, where they form cell collectives that compete for space. Within these collectives, cells often secrete molecules that benefit surface spreading by, for example, reducing surface tension or promoting filamentous growth. Although we have a detailed understanding of how these molecules are produced, much remains unknown about their role in surface competition. Here we examine sliding motility in Bacillus subtilis and compare how secreted molecules, essential for sliding, affect intraspecific cooperation and competition on a surface. We specifically examine (i) the lipopeptide surfactin, (ii) the hydrophobin protein BslA, and (iii) exopolysaccharides (EPS). We find that these molecules have a distinct effect on surface competition. Whereas surfactin acts like a common good, which is costly to produce and benefits cells throughout the surface, BslA and EPS are cost-free and act locally. Accordingly, surfactin deficient mutants can exploit the wild-type strain in competition for space, while BslA and EPS mutants cannot. Supported by a mathematical model, we show that three factors are important in predicting the outcome of surface competition: the costs of molecule synthesis, the private benefits of molecule production, and the diffusion rate. Our results underscore the intricate extracellular biology that can drive bacterial surface competition.

Computational pipeline for designing guide RNAs for mismatch-CRISPRi.

CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020).

Cryptic surface-associated multicellularity emerges through cell adhesion and its regulation.

The repeated evolution of multicellularity led to a wide diversity of organisms, many of which are sessile, including land plants, many fungi, and colonial animals. Sessile organisms adhere to a surface for most of their lives, where they grow and compete for space. Despite the prevalence of surface-associated multicellularity, little is known about its evolutionary origin. Here, we introduce a novel theoretical approach, based on spatial lineage tracking of cells, to study this origin. We show that multicellularity can rapidly evolve from two widespread cellular properties: cell adhesion and the regulatory control of adhesion. By evolving adhesion, cells attach to a surface, where they spontaneously give rise to primitive cell collectives that differ in size, life span, and mode of propagation. Selection in favor of large collectives increases the fraction of adhesive cells until a surface becomes fully occupied. Through kin recognition, collectives then evolve a central-peripheral polarity in cell adhesion that supports a division of labor between cells and profoundly impacts growth. Despite this spatial organization, nascent collectives remain cryptic, lack well-defined boundaries, and would require experimental lineage tracking technologies for their identification. Our results suggest that cryptic multicellularity could readily evolve and originate well before multicellular individuals become morphologically evident.

Short-range quorum sensing controls horizontal gene transfer at micron scale in bacterial communities.

In bacterial communities, cells often communicate by the release and detection of small diffusible molecules, a process termed quorum-sensing. Signal molecules are thought to broadly diffuse in space; however, they often regulate traits such as conjugative transfer that strictly depend on the local community composition. This raises the question how nearby cells within the community can be detected. Here, we compare the range of communication of different quorum-sensing systems. While some systems support long-range communication, we show that others support a form of highly localized communication. In these systems, signal molecules propagate no more than a few microns away from signaling cells, due to the irreversible uptake of the signal molecules from the environment. This enables cells to accurately detect micron scale changes in the community composition. Several mobile genetic elements, including conjugative elements and phages, employ short-range communication to assess the fraction of susceptible host cells in their vicinity and adaptively trigger horizontal gene transfer in response. Our results underscore the complex spatial biology of bacteria, which can communicate and interact at widely different spatial scales.

A riboswitch gives rise to multi-generational phenotypic heterogeneity in an auxotrophic bacterium.

Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.

Microbial life cycles link global modularity in regulation to mosaic evolution.

Microbes are exposed to changing environments, to which they can respond by adopting various lifestyles such as swimming, colony formation or dormancy. These lifestyles are often studied in isolation, thereby giving a fragmented view of the life cycle as a whole. Here, we study lifestyles in the context of this whole. We first use machine learning to reconstruct the expression changes underlying life cycle progression in the bacterium Bacillus subtilis, based on hundreds of previously acquired expression profiles. This yields a timeline that reveals the modular organization of the life cycle. By analysing over 380 Bacillales genomes, we then show that life cycle modularity gives rise to mosaic evolution in which life stages such as motility and sporulation are conserved and lost as discrete units. We postulate that this mosaic conservation pattern results from habitat changes that make these life stages obsolete or detrimental. Indeed, when evolving eight distinct Bacillales strains and species under laboratory conditions that favour colony growth, we observe rapid and parallel losses of the sporulation life stage across species, induced by mutations that affect the same global regulator. We conclude that a life cycle perspective is pivotal to understanding the causes and consequences of modularity in both regulation and evolution.

Emergence of diverse life cycles and life histories at the origin of multicellularity.

The evolution of multicellularity has given rise to a remarkable diversity of multicellular life cycles and life histories. Whereas some multicellular organisms are long-lived, grow through cell division, and repeatedly release single-celled propagules (for example, animals), others are short-lived, form by aggregation, and propagate only once, by generating large numbers of solitary cells (for example, cellular slime moulds). There are no systematic studies that explore how diverse multicellular life cycles can come about. Here, we focus on the origin of multicellularity and develop a mechanistic model to examine the primitive life cycles that emerge from a unicellular ancestor when an ancestral gene is co-opted for cell adhesion. Diverse life cycles readily emerge, depending on ecological conditions, group-forming mechanism, and ancestral constraints. Among these life cycles, we recapitulate both extremes of long-lived groups that propagate continuously and short-lived groups that propagate only once, with the latter type of life cycle being particularly favoured when groups can form by aggregation. Our results show how diverse life cycles and life histories can easily emerge at the origin of multicellularity, shaped by ancestral constraints and ecological conditions. Beyond multicellularity, this finding has similar implications for other major transitions, such as the evolution of sociality.

When We Stop Thinking about Microbes as Cells.

Microbes are often thought of as individual cells. However, in their natural habitats, they typically exist in the context of other cells, be they of the same or different species. How these cells interact in space and time is key to their ecology and evolution. In this perspective, we consider the implications of this in terms of the future of microbiological research. This article is part of the special issue "Microbiology: how to bridge mechanisms and phenomenology" edited by Suckjoon Jun.

PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis.

Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing ß-1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic.

On the origin of biological construction, with a focus on multicellularity.

Biology is marked by a hierarchical organization: all life consists of cells; in some cases, these cells assemble into groups, such as endosymbionts or multicellular organisms; in turn, multicellular organisms sometimes assemble into yet other groups, such as primate societies or ant colonies. The construction of new organizational layers results from hierarchical evolutionary transitions, in which biological units (e.g., cells) form groups that evolve into new units of biological organization (e.g., multicellular organisms). Despite considerable advances, there is no bottom-up, dynamical account of how, starting from the solitary ancestor, the first groups originate and subsequently evolve the organizing principles that qualify them as new units. Guided by six central questions, we propose an integrative bottom-up approach for studying the dynamics underlying hierarchical evolutionary transitions, which builds on and synthesizes existing knowledge. This approach highlights the crucial role of the ecology and development of the solitary ancestor in the emergence and subsequent evolution of groups, and it stresses the paramount importance of the life cycle: only by evaluating groups in the context of their life cycle can we unravel the evolutionary trajectory of hierarchical transitions. These insights also provide a starting point for understanding the types of subsequent organizational complexity. The central research questions outlined here naturally link existing research programs on biological construction (e.g., on cooperation, multilevel selection, self-organization, and development) and thereby help integrate knowledge stemming from diverse fields of biology.

Regulatory mechanisms link phenotypic plasticity to evolvability.

Organisms have a remarkable capacity to respond to environmental change. They can either respond directly, by means of phenotypic plasticity, or they can slowly adapt through evolution. Yet, how phenotypic plasticity links to evolutionary adaptability is largely unknown. Current studies of plasticity tend to adopt a phenomenological reaction norm (RN) approach, which neglects the mechanisms underlying plasticity. Focusing on a concrete question - the optimal timing of bacterial sporulation - we here also consider a mechanistic approach, the evolution of a gene regulatory network (GRN) underlying plasticity. Using individual-based simulations, we compare the RN and GRN approach and find a number of striking differences. Most importantly, the GRN model results in a much higher diversity of responsive strategies than the RN model. We show that each of the evolved strategies is pre-adapted to a unique set of unseen environmental conditions. The regulatory mechanisms that control plasticity therefore critically link phenotypic plasticity to the adaptive potential of biological populations.

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles.

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a 'sticky' cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles.

Division of Labor in Biofilms: the Ecology of Cell Differentiation.

The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental conditions, but they also differentiate into cell types that interact with each other. This allows for task differentiation and, hence, the division of labor. In this article, we focus on cell differentiation and the division of labor in three bacterial species: Myxococcus xanthus, Bacillus subtilis, and Pseudomonas aeruginosa. During biofilm formation each of these species differentiates into distinct cell types, in some cases leading to cooperative interactions. The division of labor and the cooperative interactions between cell types are assumed to yield an emergent ecological benefit. Yet in most cases the ecological benefits have yet to be elucidated. A notable exception is M. xanthus, in which cell differentiation within fruiting bodies facilitates the dispersal of spores. We argue that the ecological benefits of the division of labor might best be understood when we consider the dynamic nature of both biofilm formation and degradation.

From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed.

Bet-hedging during bacterial diauxic shift.

When bacteria grow in a medium with two sugars, they first use the preferred sugar and only then start metabolizing the second one. After the first exponential growth phase, a short lag phase of nongrowth is observed, a period called the diauxie lag phase. It is commonly seen as a phase in which the bacteria prepare themselves to use the second sugar. Here we reveal that, in contrast to the established concept of metabolic adaptation in the lag phase, two stable cell types with alternative metabolic strategies emerge and coexist in a culture of the bacterium Lactococcus lactis. Only one of them continues to grow. The fraction of each metabolic phenotype depends on the level of catabolite repression and the metabolic state-dependent induction of stringent response, as well as on epigenetic cues. Furthermore, we show that the production of alternative metabolic phenotypes potentially entails a bet-hedging strategy. This study sheds new light on phenotypic heterogeneity during various lag phases occurring in microbiology and biotechnology and adjusts the generally accepted explanation of enzymatic adaptation proposed by Monod and shared by scientists for more than half a century.