Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B. subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.

Bacillus subtilis extracellular protease production incurs a context-dependent cost.

Microbes encounter a wide range of polymeric nutrient sources in various environmental settings, which require processing to facilitate growth. Bacillus subtilis, a bacterium found in the rhizosphere and broader soil environment, is highly adaptable and resilient due to its ability to utilise diverse sources of carbon and nitrogen. Here, we explore the role of extracellular proteases in supporting growth and assess the cost associated with their production. We provide evidence of the essentiality of extracellular proteases when B. subtilis is provided with an abundant, but polymeric nutrient source and demonstrate the extracellular proteases as a shared public good that can operate over a distance. We show that B. subtilis is subjected to a public good dilemma, specifically in the context of growth sustained by the digestion of a polymeric food source. Furthermore, using mathematical simulations, we uncover that this selectively enforced dilemma is driven by the relative cost of producing the public good. Collectively, our findings reveal how bacteria can survive in environments that vary in terms of immediate nutrient accessibility and the consequent impact on the population composition. These findings enhance our fundamental understanding of how bacteria respond to diverse environments, which has importance to contexts ranging from survival in the soil to infection and pathogenesis scenarios.

Mechanisms driving spatial distribution of residents in colony biofilms: an interdisciplinary perspective.

Biofilms are consortia of microorganisms that form collectives through the excretion of extracellular matrix compounds. The importance of biofilms in biological, industrial and medical settings has long been recognized due to their emergent properties and impact on surrounding environments. In laboratory situations, one commonly used approach to study biofilm formation mechanisms is the colony biofilm assay, in which cell communities grow on solid-gas interfaces on agar plates after the deposition of a population of founder cells. The residents of a colony biofilm can self-organize to form intricate spatial distributions. The assay is ideally suited to coupling with mathematical modelling due to the ability to extract a wide range of metrics. In this review, we highlight how interdisciplinary approaches have provided deep insights into mechanisms causing the emergence of these spatial distributions from well-mixed inocula.

Systematic microscopical analysis reveals obligate synergy between extracellular matrix components during Bacillus subtilis colony biofilm development.

Single-species bacterial colony biofilms often present recurring morphologies that are thought to be of benefit to the population of cells within and are known to be dependent on the self-produced extracellular matrix. However, much remains unknown in terms of the developmental process at the single cell level. Here, we design and implement systematic time-lapse imaging and quantitative analyses of the growth of Bacillus subtilis colony biofilms. We follow the development from the initial deposition of founding cells through to the formation of large-scale complex structures. Using the model biofilm strain NCIB 3610, we examine the movement dynamics of the growing biomass and compare them with those displayed by a suite of otherwise isogenic matrix-mutant strains. Correspondingly, we assess the impact of an incomplete matrix on biofilm morphologies and sessile growth rate. Our results indicate that radial expansion of colony biofilms results from the division of bacteria at the biofilm periphery rather than being driven by swelling due to fluid intake. Moreover, we show that lack of exopolysaccharide production has a negative impact on cell division rate, and the extracellular matrix components act synergistically to give the biomass the structural strength to produce aerial protrusions and agar substrate-deforming ability.

Founder cell configuration drives competitive outcome within colony biofilms.

Bacteria can form dense communities called biofilms, where cells are embedded in a self-produced extracellular matrix. Exploiting competitive interactions between strains within the biofilm context can have potential applications in biological, medical, and industrial systems. By combining mathematical modelling with experimental assays, we reveal that spatial structure and competitive dynamics within biofilms are significantly affected by the location and density of the founder cells used to inoculate the biofilm. Using a species-independent theoretical framework describing colony biofilm formation, we show that the observed spatial structure and relative strain biomass in a mature biofilm comprising two isogenic strains can be mapped directly to the geographical distributions of founder cells. Moreover, we define a predictor of competitive outcome that accurately forecasts relative abundance of strains based solely on the founder cells' potential for radial expansion. Consequently, we reveal that variability of competitive outcome in biofilms inoculated at low founder density is a natural consequence of the random positioning of founding cells in the inoculum. Extension of our study to non-isogenic strains that interact through local antagonisms, shows that even for strains with different competition strengths, a race for space remains the dominant mode of competition in low founder density biofilms. Our results, verified by experimental assays using Bacillus subtilis, highlight the importance of spatial dynamics on competitive interactions within biofilms and hence to related applications.

Translational control of gene expression via interacting feedback loops.

Translation is a key step in the synthesis of proteins. Accordingly, cells have evolved an intricate array of control mechanisms to regulate this process. By constructing a multicomponent mathematical framework we uncover how translation may be controlled via interacting feedback loops. Our results reveal that this interplay gives rise to a remarkable range of protein synthesis dynamics, including oscillations, step change, and bistability. This suggests that cells may have recourse to a much richer set of control mechanisms than was previously understood.

Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm.

Biofilm formation by Bacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm into a nonexpanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that B. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe3+ from the growth medium in a nonenzymatic reaction. Utilizing mathematical modeling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated with pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion by neighboring communities through the generation of an "iron-free" zone, thereby addressing the paradox of pulcherriminic acid production by B. subtilis.

Auto-aggregation in zoospores of Phytophthora infestans: the cooperative roles of bioconvection and chemotaxis.

Phytophthora infestans is a highly destructive plant pathogen. It was the cause of the infamous Irish potato famine in the nineteenth century and remains to this day a significant global problem with associated costs estimated at $3 billion annually. Key to the success of this pathogen is the dispersal of free-swimming cells called zoospores. A poorly understood aspect of zoospore behaviour is auto-aggregation--the spontaneous formation of large-scale patterns in cell density. Current competing hypotheses suggest that these patterns are formed by one of two distinct mechanisms: chemotaxis and bioconvection. In this paper, we present mathematical and experimental results that together provide strong evidence that auto-aggregation can only result from a combination of these mechanisms, each having a distinct, time-separated role. A better understanding of the underlying infection mechanisms of P. infestans and potentially other Phytophthora species will in the longer term lead to advances in preventative treatment and thus potentially significant savings in socio-economic costs.

Regulation of vectorial supply of vesicles to the hyphal tip determines thigmotropism in Neurospora crassa.

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip.

Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm.

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

A synthetic system for expression of components of a bacterial microcompartment.

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Selective heterogeneity in exoprotease production by Bacillus subtilis.

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical "bet-hedging" behaviour.

Reversing invasion in bistable systems.

In this paper, we discuss a class of bistable reaction-diffusion systems used to model the competitive interaction of two species. The interactions are assumed to be of classic "Lotka-Volterra" type and we will consider a particular problem with relevance to applications in population dynamics: essentially, we study under what conditions the interplay of relative motility (diffusion) and competitive strength can cause waves of invasion to be halted and reversed. By establishing rigorous results concerning related degenerate and near-degenerate systems, we build a picture of the dependence of the wave speed on system parameters. Our results lead us to conjecture that this class of competition model has three "zones of response". In the central zone, varying the motility can slow, halt and reverse invasion. However, in the two outer zones, the direction of invasion is independent of the relative motility and is entirely determined by the relative competitive strengths. Furthermore, we conjecture that for a large class of competition models of the type studied here, the wave speed is an increasing function of the relative motility.

Mathematical modelling of fungal growth and function.

This contribution is based on the six presentations given at the Special Interest Group meeting on Mathematical modelling of fungal growth and function held during IMC9. The topics covered aspects of fungal growth ranging across several orders of magnitude of spatial and temporal scales from the bio-mechanics of spore ejection, vesicle trafficking and hyphal tip growth to the form and function of mycelial networks. Each contribution demonstrated an interdisciplinary approach to questions at specific scales. Collectively, they represented a significant advance in the multi-scale understanding of fungal biology.

An integrated pharmacokinetic-pharmacodynamic model for an Aurora kinase inhibitor.

The spindle assembly checkpoint is a cell cycle surveillance mechanism that ensures the proper separation of chromosomes prior to cell division at mitosis. Aurora kinases play critical roles in mitotic progression and hence small-molecule inhibitors of Aurora kinases have been developed as a new class of potential anti-cancer drugs. In this paper we present for the first time an integrated pharmacokinetic-pharmacodynamic model of the functional effects of CYC116 (a known inhibitor of Aurora kinases A and B) on the spindle assembly checkpoint. We use the model to simulate two common experimental systems: cell culture and p.o. dosing of mice and present predictions of the effects of CYC116 for a range of doses and drug scheduling regimes. The model reveals that a critical peak drug concentration is required to cause aberrant kinetochore-microtubule attachments. The model also predicts that provided this threshold concentration is exceeded, a high total oral dose causes a high number of aberrant attachments within any given damaged cell. However, the proportion of cells which enter anaphase with aberrant attachments is associated with the total length of time for which the plasma concentration is maintained above the threshold. Moreover, our model reveals that the length of prometaphase/metaphase is a nonlinear function of drug dose and this time period can be extended or shortened. Finally, a strong saturation effect on CYC116 efficacy is predicted by the model. We discuss how these predictions may have implications for further drug trials using CYC116 and other similar AK inhibitors.

A pharmacodynamic model of Aurora kinase inhibitors in the spindle assembly checkpoint.

Arguably the most dramatic phase in the cell cycle is mitosis, during which replicated chromosomes are sorted into two distinct sets. Aurora kinases are central to the accurate segregation of chromosomes during mitosis. Consequently, they have been selected as possible targets for cancer therapy. Anti-cancer drugs that target Aurora kinases are normally designed to inhibit their function. The complexity of the roles of Aurora kinases and their interaction with respective inhibitors means that it is often very difficult to obtain meaningful links between inhibitor concentration and efficacy using standard methods. To overcome these difficulties, we propose a novel mathematical modelling approach. We present a pharmacodynamic model that is able to encapsulate the key roles of two kinases, Aurora A and B, in the spindle assembly checkpoint. Moreover, the model is capable of qualitatively differentiating between the effects of inhibiting Aurora A, Aurora B and A plus B, respectively, by predicting cell behaviour. Consequently, predictions regarding the qualitative relationship between inhibitors, measurable biomarkers and cell damage can be obtained using this powerful modelling approach.

Modeling the temporal evolution of the spindle assembly checkpoint and role of Aurora B kinase.

Faithful separation of chromosomes prior to cell division at mitosis is a highly regulated process. One family of serine/threonine kinases that plays a central role in regulation is the Aurora family. Aurora B plays a role in the spindle assembly checkpoint, in part, by destabilizing the localization of BubR1 and Mad2 at centrosomes and responds to changes in tension caused by aberrant microtubule kinetochore attachments. Aurora B is overexpressed in a subset of cancers and is required for mitosis, making it an attractive anticancer target. Here, we use mathematical modeling to extend a current model of the spindle assembly checkpoint to incorporate all signaling kinetochores within a cell rather than just one and the role of Aurora B within the resulting model. We find that the current model of the spindle assembly checkpoint is robust to variation in its key diffusion-limited parameters. Furthermore, when Aurora B inhibition is considered within the model, for a certain range of inhibitor concentrations, a prolonged prometaphase/metaphase is observed. This level of inhibitor concentrations has not yet been studied experimentally, to the authors' best knowledge. Therefore, experimental verification of the results discussed here could provide a deeper understanding of how kinetochores and Aurora B cooperate in the spindle assembly checkpoint.

Induction of contour sensing in Aspergillus niger by stress and its relevance to fungal growth mechanics and hyphal tip structure.

Thigmotropism (contour sensing) has been assigned an important role in both plant and human fungal pathogens. However, outside these systems, our knowledge of the function of thigmotropism in fungal growth control is relatively poor. Furthermore, the effects of environmental stress on thigmotropic responses have received scant attention. To try to elucidate some of the mechanisms behind hyphal contour sensing in response to nutrient-poor environments, we have used micro-engineered substrates and several imaging techniques to investigate the thigmotropic reactions of the ubiquitous fungus Aspergillus niger. This organism not appear to demonstrate thigmotropic growth under normal conditions. Our results show that A. niger undergoes significant morphological changes during growth on solid substrates and demonstrate that the intensity of contour sensing varies depending on the area of the hyphal tip which initiates contact with the substrate. We propose that growth under nutrient-limited conditions triggers several factors that combine to increase thigmotropic sensitivity while conversely creating a 60 degrees arc at the hyphal tip which is blind to topographical variations. This has important consequences for our general understanding of the hyphal mode of growth in fungi as well as more specific aspects of hyphal tip development under stress.