Interdisciplinary life of microbes: from single cells to multicellular aggregates

In wild environments, mixtures of motile and sessile bacterial species interact physically and chemically to give rise to complex community organization. We investigated the little studied role of physical interactions in this organization. When two otherwise identical strains of E coli, one motile and the other not, are mixed, we observe, in a physiologically relevant range of densities, the emergence of large scale fluctuations of the density of the non-motile strain, driven by the swimming activity of the motile cells. We systematically investigated the phase diagram of this phenomenon and identified some essential physical ingredients for its emergence: the localization of the swimmers at the surfaces of the experimental chamber, which creates a spatiotemporally organized active noise on the non-motile cells (via hydrodynamic interactions), and the collective sedimentation of the swimmers. They together induce a convective behavior that generates the density heterogeneities. These density fluctuations appear to have strong implications for the structure and dynamics of formation of cellular aggregates of the non-motile cells and should therefore strongly influence the structure of multispecies biofilms.

Natural isolates of the endospore-forming model bacterium Bacillus subtilis show complex multicellular traits. Similar to plants, out of a spore of B. subtilis a 3D macroscopic multicellular structure can emerge that produces many more spores - a fruiting body. Different cell types partake in the formation of a fruiting body and its substructures. We will outline our approach to decipher how cell-type specific functions support biofilm formation using a combination of different imaging technqiues and computer-based image analysis methods. With this approach we want to test different hypotheses regarding the formation of fruiting bodies by employing targeted genetic manipulations of B. subtilis to reveal whether and how these genetic elements contribute to emergent properties during multicellular development.

Alexander Balinovic1,2,3, Stephan Wimmi4, Bartosz Turkowyd 1,3, Andreas Diepold 4, Ulrike Endesfelder 1,2,3 1 Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany 2 Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, US 3 Institute for Microbiology and Biotechnology, Rheinische Friedrich Wilhelm Universität, Bonn, Germany (current address) 4 Max Planck Institute for Terrestrial Microbiology, Ecophysiology, Marburg, Germany Yersinia enterocolitica is a gastrointestinal human pathogen that uses a type III secretion system (T3SS), also called injectisome, as a major virulence factor to manipulate host cell behavior to its benefits [1]. Upon host cell contact, effector proteins from the bacterial cytoplasm are secreted into the host cytoplasm via the T3SS, which establishes a direct connection between the bacteria and the host cell [2-4]. Although the structure of the T3SS is well understood and can be divided into membrane-bound and cytosolic components, it is remarkably less known about the molecular regulation and dynamics of the T3SS components. In the first project [1], we investigated the molecular regulation of the T3SS activity and found that external pH environments have a strong influence on the mobility of the cytosolic components of the T3SS. We could show that at low external pH, effector secretion is inhibited by temporarily release of the cytosolic components. The membrane component SctD was shown to be responsible for sensing the drop in external pH and partially dissociating from the membrane. This process was shown to be reversible upon restoration of neutral pH. These results strongly indicate a regulatory mechanism, which controls T3SS activity in response to changes in environmental conditions, e.g. the different pH environments Y. enterocolitica encounters during its infection route through the gastrointestinal tract. In a second project [5], we aimed to understand the recruitment of effector proteins by the sorting platform, a conserved complex consisting of cytosolically diffusing proteins, and the dynamics of these effector-sorting platform complexes. We found that the sorting platform proteins were part of ~5.1 and ~17.9 injectisomes per bacterium under non-secreting and secreting conditions, respectively. Furthermore, they displayed a considerable mobile cytosolic pool with effector proteins directly binding to the proteins SctQ and SctL within the cytosol. In wild-type bacteria, these proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these complexes is modulated by the presence of effector proteins and their chaperones, and upon initiation of secretion, which additionally leads to the emergence of large soluble complexes. Our quantitative data supports an effector shuttling mechanism, in which sorting platform protein bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome. [1] Wimmi et al., Nature Comm., 2021, [2] Costa et al., Nature Rev. Microbiol., 2015, [3] Green & Mecsas, Microbiol. Spect., 2020, [4] Denise et al., Trends Microbiol., 2020, [5] Wimmi et al., manuscript submitted

Biofilms are heterogeneous bacterial communities that are held together and stick to surfaces by a self-produced extracellular matrix (ECM). Biofilms may be compared with multicellular organisms where cells organize into sub-groups with different functions. Bacterial biofilms also respond to damage and heal their wounds, however, the mechanism underlying this process is not yet resolved. In our study, we combine biophysical experiments and theoretical modeling to characterize the wound healing process and better understand the role of biofilm components (cells, ECM), aging, and nutrient consumption on wound closure. Using Bacillus subtilis as a model organism for biofilm formation, we quantified the changes in the biofilm biomass and matrix production along time during the wound closure. Combining biophysical tools such as AFM, optical, and electron microscopy with computational modeling, we will determine the parameters that are responsible for wound healing and quantify changes in biomass, forces, and mechanical properties across the healing region. This study is an important milestone in the comparison between bacterial biofilms and tissues.

Membrane potential in bacterial systems has been shown to be dynamic and tightly related to survivability at the single cell level. However, little is known about spatio-temporal patterns of membrane potential in bacterial colonies and biofilms. We discovered a switch from uncorrelated to collective dynamics within colonies formed by the human pathogen Neisseria gonorrhoeae. In freshly assembled colonies, polarization is heterogeneous with instances of transient and uncorrelated hyper- or depolarization of individual cells. As colonies reach a critical size, the polarization behaviour switches to collective dynamics: A hyperpolarized shell forms at the centre, travels radially outward, and halts several micrometres from the colony periphery. Once the shell has passed, we detect an influx of potassium correlated with depolarisation. Transient hyperpolarization also demarks the transition from volume to surface growth. By combining simulations and the use of an alternative electron acceptor for the respiratory chain, we provide strong evidence that local oxygen gradients shape the collective polarization dynamics. Finally, we show that within the hyperpolarized shell, tolerance against aminoglycoside antibiotics but not against β-lactam antibiotics is increased, suggesting that depolarization instantaneously protects cells, while the protective effect of growth arrest does not set in immediately. These findings highlight that the polarization pattern can demark the differentiation into distinct subpopulations with different growth rates and antibiotic tolerance.

Recent research shows that in confined populations of growing and dividing rods, such as microcolonies of bacteria, a complex interplay between growth activity, fluctuating inter-particle forces and boundary effects can lead to emergent collective dynamics, including global flow of cellular matter and alignment due to the nematic symmetry of local mechanical interactions. Here, we use a new versatile framework for agent-based simulations to explore these effects in systems with different geometries containing two-dimensional spherocylinders. We observe the emergence of orientational order in rectangular channels and analyse its dependence on both microscopic parameters of the rods and the geometry of the confinement. Further observations of complex orientation patterns in open polygonal domains hint at a link between shear rate anisotropy and orientation.

A central feature of living matter is its ability to grow and multiply. The mechanical activity associated with growth produces both macroscopic flows shaped by confinement, and striking self-organization phenomena, such as orientational order and alignment, which are particularly prominent in populations of rod-shaped bacteria due to their nematic properties. However, how active stresses, passive mechanical interactions and flow-induced effects interact to give rise to the observed global alignment patterns remains elusive. We simulate colonies of growing rod-shaped particles of different aspect ratios confined in channel-like geometries. A spatially resolved analysis of the stress tensor reveals a strong relationship between near-perfect alignment and an inversion of stress anisotropy for particles with large length-to-width ratios. We show that, in quantitative agreement with an asymptotic theory, strong alignment can lead to a decoupling of active and passive stresses parallel and perpendicular to the direction of growth, respectively. We demonstrate the robustness of these effects in a geometry that provides less restrictive confinement and introduces natural perturbations in alignment. Our results illustrate the complexity arising from the inherent coupling between nematic order and active stresses in growing active matter which is modulated by geometric and configurational constraints due to confinement.

In nature, bacterial collectives typically consist of multiple species, which are interacting both biochemically and physically. Nonetheless, past studies on the physical properties of swarming bacteria were focused on axenic (single-species) populations. In bacterial swarming, intricate interactions between the individuals lead to clusters, rapid jets, and vortices that depend on cell characteristics such as speed and length. In this work, we show the first results of rapidly swarming mixed-species populations of Bacillus subtilis and Serratia marcescens, two model swarm species that are known to swarm well in axenic situations. In mixed liquid cultures, both species have higher reproduction rates. We show that the mixed population swarms together well and that the fraction between the species determines all dynamical scales—from the microscopic (e.g., speed distribution), mesoscopic (vortex size), and macroscopic (colony structure and size). Understanding mixed-species swarms is essential for a comprehensive understanding of the bacterial swarming phenomenon and its biological and evolutionary implications.

Phocaeicola (Bacteroides) vulgatus (Pvu) is a common member of the human gut microbiota and is associated with cardiometabolic disease and inflammatory bowel disease. While Pvu is an effective gut colonizer, fundamental gaps remain in our knowledge of the mechanisms that contribute to its colonization and association with inflammatory phenotypes. Our goal was to leverage insertion sequencing (INseq), murine colonization studies, and metabolomics to identify key metabolites that promote Pvu gut colonization. A murine colonization screen using a pooled transposon library of Pvu was used to identify genes required for in vivo survival when either alone or competition with native mouse microbiota. We subsequently prioritized mutants with altered colonization outcomes for untargeted metabolomics. INseq mutant 777 was particularly interesting due to its depletion in the gut when colonizing specific-pathogen-free mice but its enrichment when competing against only other Pvu strains in the absence of native gut microbiota. Untargeted metabolomics indicated that multiple Pvu INseq mutants demonstrated dramatic metabolomic differences from WT when cultivated on agar. In particular, 777 had a metabolome distinct from WT, producing increased levels of lipid metabolites. Genetic analysis determined that 777 harbors an insertion in a putative lipid transport operon unique to Pvu and closely related bacteria. Ongoing work aims to characterize the genes and metabolic products of this operon in vitro using clean deletion mutants and additional mouse studies. Additional analyses comparing the metabolomes of other INseq mutants to WT are underway. Overall, this study bolsters our understanding of Pvu genetics and metabolism, as well as the potential mechanisms underlying bacterial gut colonization, insights that could be leveraged to develop therapeutics to treat Pvu-related diseases or alter gut microbial ecology.

A mathematical model for the interacting dynamics of phytoplankton-zooplankton is proposed. The phytoplankton have ability to take refuge and release toxins to avoid over predation by zooplankton. The zooplankton are provided some additional food to persist in the system. The phytoplankton are assumed to be affected directly by an external toxic substance whereas zooplankton are affected indirectly by feeding on the affected phytoplankton. We incorporate seasonal variations in the model, assuming the level of nutrients, refuge and the rate of toxins released by phytoplankton as functions of time. Our results show that when high toxicity and refuge cause extinction of zooplankton, providing additional food supports the survival of zooplankton population and controls the phytoplankton population. Prey refuge and additional food have stabilizing effects on the system; higher values of the former results in extinction of zooplankton whereas phytoplankton disappear for larger values of the latter. Seasonality in nutrients level and toxins released by phytoplankton generates higher periodic solutions while time-dependent refuge of phytoplankton causes the occurrence of a period-three solution. The possibility of finding additional food for zooplankton may push back the ecosystem to a simple stable state from a complex dynamics.

Curli amyloid fibers are the key component of the biofilms produced by E. coli at low temperatures (30 degrees Celsius and lower). Curli expression is under the control of stationary phase sigma S factor and c-di-GMP regulatory network. Using fluorescence reporter of curli gene expression combined with flow cytometry analysis, we show that bimodal activation of curli expression occurs not only in submerged and macrocolony biofilms, but also in well-mixed planktonic cultures of E. coli upon the entry into stationary growth phase. It leads to stochastic differentiation of cell population into two distinct subpopulations of curli-positive and curli-negative cells. Such differentiation is also observed on the single-cell level when cells are grown in the conditioned medium in microfluidics device and this curli-positive state is only metastable. Curli expression is strongly reduced when E. coli cultures are grown in a concentrated TB medium or with addition of serine and strongly enhanced in the presence of serine hydroxamate, which all affect stringent response in cells. With the help of gene knock-out approach we further show that regulation of curli expression by c-di-GMP modulates the fraction of curli-positive cells under all tested growth conditions. Notably, when c-di-GMP regulation of curli expression is abolished, the fraction of curli-positive cells shows stronger dependence on the growth conditions. We also conclude that c-di-GMP regulatory network is apparently not the origin of bimodal activation in E. coli.

Adrien Sarlet(1), Ricardo Ziege(1), Kerstin G. Blank(2,3), Peter Fratzl(1), Cécile M. Bidan(1) (1)Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam, Germany (2)Max Planck Institute of Colloids and Interfaces, Mechano(bio)chemistry, Potsdam, Germany (3)Johannes Kepler University, Institute of Experimental Physics, Linz, Austria The material properties of biofilms depend on the composition and microstructure of their extracellular polymeric substance (EPS). In particular, E. coli biofilms present tissue-like elasticity, originating from a dense fibre network made of amyloid curli and phosphoethanolamine-modified cellulose (pEtN-cellulose) [1,2]. Here, we used a set of E. coli mutants [3,4] to investigate the effect of EPS composition on E. coli biofilm morphology, texture and mechanical properties. Furthermore, to probe the influence of EPS architecture, we compared the influence of sample homogenization on the local and global mechanical biofilm properties. Microindentation of as-grown biofilms showed that biofilms expressing both matrix components present rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, biofilms lacking curli fibres are softer by one order of magnitude, but yield much higher adhesive energies. This suggests that curli and pEtN-cellulose fibres form a composite material where curli fibres provide rigidity and pEtN-cellulose serves as a glue. Oscillatory rheology of homogenized biofilms did not reveal the respective contributions of the two matrix components. All storage moduli were in the range of tens of kPa, irrespective of matrix composition. This suggests that biofilm homogenization leads to a partial destruction of the matrix architecture. Microindentation of homogenized versus as-grown biofilms showed that the rigidity decreased by a factor 4 for homogenized biofilms containing both types of fibres. Overall, these results confirm that both EPS composition and architecture determine biofilm mechanical properties. [1] Serra, D. O., Klauck, G., & Hengge, R. (2015) Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli. Environmental Microbiology 17:5073 [2] Jeffries, J., Thongsomboon, W., Visser, J. A., Enriquez, K., Yager, D., & Cegelski, L. (2020) Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties. Biopolymers 112: e23395 [3] Serra, D. O., Richter, A. M., & Hengge, R. (2013).Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms. Journal of Bacteriology 195:5540 [4] Thongsomboon, W., Serra, D. O., Possling, A., Hadjineophytou, C., Hengge, R., & Cegelski, L. (2018) Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose. Science 359:334

Bacterial populations do not usually exist as a homogenous community in nature. Successful pathogenic colonization must overcome a community of commensal bacteria that co-exists with the human body during a bacterial infection in the human body. One of the examples we are interested in this study is the interaction between two Neisseria species, N. gonorrhoeae(Ng) being the pathogen while N. elongata(Nel) is the commensal counterpart. Given the close evolutionary proximity, these two species share a lot in common. They are found in human epidermal tissues, in particular mucosas. They also both possess Type IV Pili (T4P), a ubiquitous bacterial appendage which play an essential role in adherence to human tissues and natural competence for both species. A previous study observed that co-culturing these two species leads to a deterioration of the Ng population termed ‘killing’. This information opens doors to many questions. In this study, we aim to improve the understanding of how this ‘killing’ happen. We have reproduced the same phenomenon and examined the killing dynamic in time and space. We also identified a few Ng mutants that resist this killing through genetic screening. Our current hypothesis points the project toward a few possible candidate pathways: DNA uptake, pilin interaction and cell metabolism. This meeting will play a role in providing insight for our further investigation. Since this study revolves around bacterial cells in the community and potentially in a host, several topics covered in the workshop will help enlighten us on better tools or angles for future work. These include talks on bacterial growth, metabolism and fitness, and bacteria infection or pathogenesis.

Biofilms, or approximately two-dimensional microbial colonies on the surface, are ubiquitous both in natural and industrial settings. They also present an excellent case of evolution in the ecological community of interacting organisms, which is relatively easy to study in the lab environment. The dynamics of biofilms strongly depend on the mechanical interactions between individual cells. On a larger scale, these mechanical interactions lead to three essential properties of the biofilm: elastic on short time scales, viscous on large time scales, and growing. We construct a dynamic model of a viscoelastic growing fluid that incorporates all three of these properties. To validate this model, we compare it with the experiment [Kayser et al., Nature ecology & evolution, 3(1):125–134, 2019] on the growth of the biofilm containing the mixture of two strains with different growth speed, and with the KPZ equation of the boundary growth, previously used to describe this system [Kayser et al.,2019].

Bacteria can experience a rapid and huge fluctuation in intracellular c-di-GMP levels when they encounter varying environment. The triggered c-di-GMP signaling coordinates gene expression and bacterial behaviors for environmental adaptation. To accurately track c-di-GMP dynamics in cells under diverse conditions, we need a toolbox containing biosensors with different affinities for detection of c-di-GMP in vivo. We constructed 90 FRET-based fluorescent biosensors using homologues of c-di-GMP-binding effector YcgR from a set of bacterial species. Confirmed by the absolute measurements of FRET efficiency (E) using photobleaching fluorescence microscopy, a reliable calibration was applied to calculate E from flow cytometry data, allowing us to screen the biosensor library in a fast way. The E value of each biosensor was firstly compared in E. coli W3110 ∆pdeH and ∆dgcE strains, which have high and low levels of c-di-GMP, respectively. The biosensors showing large efficiency change, indicative of a large YcgR conformation change, were selected to further analysis of their E values in wild-type cells, which has intermediate level of c-di-GMP. The relative position of E in wild-type between ∆pdeH and ∆dgcE cells implied the affinity of biosensors, which is consistent with our affinity results measured in vitro. A toolbox of FRET-based c-di-GMP biosensors was set up, consisting of biosensors undergoing large FRET signal change and different c-di-GMP affinity. When screening the biosensor library, we found that the absence of sodium chloride increased cellular c-di-GMP levels via affecting diguanylate cyclase DgcE.

$Laura Zorzetto^1$, $Ernesto Scoppola^1$, $Emeline Raguin^1$, $Kerstin Blank^2^,^3$, $Peter Fratzl^1$, $Cécile M. Bidan^1$ 1 Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Am Mühlenberg 1 - 14476 Potsdam, Germany 2 Mechano(bio)chemistry, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1 - 14476 Potsdam, Germany 3Johannes Kepler Universität Linz, Institute of Experimental Physics, Altenberger Str. 69 - 4040 Linz, Austria Biofilms appear when bacteria synthesize and assemble extracellular matrix components after colonizing a surface. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphates and calcium carbonates. Previously, we investigated the role of alkaline phosphatase (ALP) in the precipitation of calcium phosphates in the form of hydroxyapatite in the biofilms produced by the model strain \textit{E. coli} K-12 W3110. From focused ion beam with scanning electron microscopy, we could identify both mineralized bacteria and mineralized portions of the extracellular matrix. This can indicate an interplay between different biomineralization processes related to microbial activity. With few exceptions, microbial mineralization usually results from adventitious precipitation of inorganic compounds led by their interactions with different metabolic processes [1]. Biologically induced mineralization results directly from microbial activity and tends to accumulate minerals on the surface of the bacteria (epicellular mineralization) and eventually embed them in the growing crystals [1]. Biologically influenced mineralization stems from the interaction between extracellular matrix and the surrounding environment [2]. In this context, we investigated the influence of different macromolecules in the biofilm on the formation of calcium phosphate crystals. On nutritive agar substrates inducing mineralization, we cultivated diverse \textit{E. coli} strains with different extracellular matrix composition, i.e. curli fibers, cellulose fibers, both types of fibers and none of them. We estimated crystal size using wide-angle x-ray scattering enabling to assess crystal strain at the nanoscale. Focused ion beam with scanning electron microscopy was exploited to study the crystal aggregation at a larger scale. Uncovering the influence of the biomolecules present in the extracellular matrix is an important piece of knowledge to engineer living composites. 1. Mann S (2001) Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry. Oxford University Press, New York, NY 2. Decho AW (2010) Overview of biopolymer-induced mineralization: What goes on in biofilms? Ecol Eng 36:137–144. https://doi.org/10.1016/j.ecoleng.2009.01.003