Effects of stochasticity and division of labor in toxin production on two-strain bacterial competition in Escherichia coli.

In phenotypically heterogeneous microbial populations, the decision to adopt one or another phenotype is often stochastically regulated. However, how this stochasticity affects interactions between competing microbes in mixed communities is difficult to assess. One example of such an interaction system is the competition of an Escherichia coli strain C, which performs division of labor between reproducers and self-sacrificing toxin producers, with a toxin-sensitive strain S. The decision between reproduction or toxin production within a single C cell is inherently stochastic. Here, combining experimental and theoretical approaches, we demonstrate that this stochasticity in the initial phase of colony formation is the crucial determinant for the competition outcome. In the initial phase (t < 12h), stochasticity influences the formation of viable C clusters at the colony edge. In the subsequent phase, the effective fitness differences (set primarily by the degree of division of labor in the C strain population) dictate the deterministic population dynamics and consequently competition outcome. In particular, we observe that competitive success of the C strain is only found if (i) a C edge cluster has formed at the end of the initial competition phase and (ii) the beneficial and detrimental effects of toxin production are balanced, which is the case at intermediate toxin producer fractions. Our findings highlight the importance of stochastic processes during the initial phase of colony formation, which might be highly relevant for other microbial community interactions in which the random choice between phenotypes can have long-lasting consequences for community fate.

Interference Disturbance Analysis Enables Single-Cell Level Growth and Mobility Characterization for Rapid Antimicrobial Susceptibility Testing.

To support the emerging battle against antimicrobial resistance (AMR), detection methods that allow fast and accurate antimicrobial susceptibility testing (AST) are urgently needed. The early identification and application of an appropriate antibiotic treatment leads to lower mortality rates and substantial cost savings and prevents the development of resistant pathogens. In this work, we present a diffraction-based method, which is capable of quantitative bacterial growth, mobility, and susceptibility measurements. The method is based on the temporal analysis of the intensity of a light diffraction peak, which arises due to interference at a periodic pattern of gold nanostructures. The presence of bacteria disturbs the constructive interference, leading to an intensity decrease and thus allows the monitoring of bacterial growth in very low volumes. We demonstrate the direct correlation of the decrease in diffraction peak intensity with bacterial cell number starting from single cells and show the capability for rapid high-throughput AST measurements by determining the minimum inhibitory concentration for three different antimicrobials in less than 2-3 h as well as the susceptibility in less than 30-40 min. Furthermore, bacterial mobility is obtained from short-term fluctuations of the diffraction peak intensity and is shown to decrease by a factor of 3 during bacterial attachment to a surface. This multiparameter detection method allows for rapid AST of planktonic and of biofilm-forming bacterial strains in low volumes and in real-time without the need of high initial cell numbers.

Locality of interactions in three-strain bacterial competition in E. coli.

The population dynamics that determine the composition and stability of ecosystems ultimately emerge from interactions between individual organisms. One well-studied system is the three-strain E. coli interaction of a heterogeneously toxin-producing C strain that interacts with a toxin-sensitive S and a toxin-resistant R strain. Here, we employ a multi-scale fluorescence microscopy approach, that has been proven useful in identifying previously unknown or underestimated stochastic effects in C-S competition. This approach allows us to investigate the microscopic interaction of the R strain and to quantify the role of stochastic effects in the spatially structured C-R-S interaction. We show that the early colony patterning at 12 h and at small length scales (near single cell level) is characterized by a number of microscopic variables (the number of C and R cell clusters and the area occupied by S) and is subject to random processes in positioning and toxin production. Then, in a second competition phase, mainly deterministic processes such as bacterial growth and global toxin action determine the following population dynamics. Consequently, together with environmental factors, the microscopic variables were predictive of the competition outcome. However, interactions of neighboring R and C clusters could amplify local variations. If R clusters originated near a C cell cluster, R could profit from the toxin produced by C without bearing the cost of production-a mechanism called cheating. By combining information from the micro- and macro-scale dynamics, we can estimate the distance at which the cheating interaction significantly changes to be in the order of 250 µm. In summary, after an initial phase influenced by stochastic patterning, largely deterministic growth dynamics follow, which are additionally affected by local interactions of neighboring clusters. As such, the results underline the importance of stochasticity and local effects in the context of ecological interactions.

Complex microbial systems across different levels of description.

Complex biological systems offer a variety of interesting phenomena at the different physical scales. With increasing abstraction, details of the microscopic scales can often be extrapolated to average or typical macroscopic properties. However, emergent properties and cross-scale interactions can impede naïve abstractions and necessitate comprehensive investigations of these complex systems. In this review paper, we focus on microbial communities, and first, summarize a general hierarchy of relevant scales and description levels to understand these complex systems: (1) genetic networks, (2) single cells, (3) populations, and (4) emergent multi-cellular properties. Second, we employ two illustrating examples, microbial competition and biofilm formation, to elucidate how cross-scale interactions and emergent properties enrich the observed multi-cellular behavior in these systems. Finally, we conclude with pointing out the necessity of multi-scale investigations to understand complex biological systems and discuss recent investigations.

Hierarchical Post-transcriptional Regulation of Colicin E2 Expression in Escherichia coli.

Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system's basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and reveals the importance of the specific regulatory components for the timing of toxin release.

Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms.

Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain,B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms.

Dynamics of ColicinE2 production and release determine the competitive success of a toxin-producing bacterial population.

The release of toxins is one mechanism used by bacterial species to establish dominance over competitors, but how the dynamics of toxin expression determine the competitive success of a toxin-producing population is largely unknown. Here, we investigate how the expression dynamics of ColicinE2 - a toxic bacteriocin - affect competition between toxin-producing and toxin-sensitive strains of Escherichia coli. We demonstrate that, in addition to genetic modifications in the toxin expression system, alterations of the growth medium can be used to modulate the timing of toxin production and the amount of toxin released. Thus cells that release the toxin at later times can accumulate more colicin. In experiments, we found that delaying toxin release does not significantly alter competition outcome. However, our theoretical analysis allowed us to assess the relative contributions of release time and toxin level to the competitive success of the producer strain, that might counteract each other in experiments. The results reveal that the importance of delaying toxin release lies in increasing the toxin amount. This is a more effective strategy for the toxin-producing strain than prompt discharge of the colicin. In summary, our study shows how the toxin release dynamics influence the competitive success of the toxin-producing bacterial population.

Gene expression noise in a complex artificial toxin expression system.

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.

CsrA and its regulators control the time-point of ColicinE2 release in Escherichia coli.

The bacterial SOS response is a cellular reaction to DNA damage, that, among other actions, triggers the expression of colicin - toxic bacteriocins in Escherichia coli that are released to kill close relatives competing for resources. However, it is largely unknown, how the complex network regulating toxin expression controls the time-point of toxin release to prevent premature release of inefficient protein concentrations. Here, we study how different regulatory mechanisms affect production and release of the bacteriocin ColicinE2 in Escherichia coli. Combining experimental and theoretical approaches, we demonstrate that the global carbon storage regulator CsrA controls the duration of the delay between toxin production and release and emphasize the importance of CsrA sequestering elements for the timing of ColicinE2 release. In particular, we show that ssDNA originating from rolling-circle replication of the toxin-producing plasmid represents a yet unknown additional CsrA sequestering element, which is essential in the ColicinE2-producing strain to enable toxin release by reducing the amount of free CsrA molecules in the bacterial cell. Taken together, our findings show that CsrA times ColicinE2 release and reveal a dual function for CsrA as an ssDNA and mRNA-binding protein, introducing ssDNA as an important post-transcriptional gene regulatory element.

Topographical alterations render bacterial biofilms susceptible to chemical and mechanical stress.

For the inactivation or removal of bacterial biofilms via chemical or physical processes, it is crucial to sufficiently wet the biofilm surface. However, many bacterial biofilms efficiently resist wetting by water, oil or even organic solvents. Here, we demonstrate how exposing the surface of mature biofilm colonies to concentrated ethanol, saline or glucose solutions results in topographical changes that enable their wettability. With this approach, even omniphobic biofilm colonies become wettable towards aqueous solutions and oils. As a result of this reduced liquid repellency, the biofilms become susceptible to erosion by water which allows for their removal from the substrate they have been grown on. Moreover, bacteria within pre-treated biofilms can now be inactivated with antibiotic solutions. Thus, the biofilm treatment strategy presented here presents a new stepping stone for fighting biofilms in either industrial or medical settings.

Modulation of the mechanical properties of bacterial biofilms in response to environmental challenges.

Bacterial communities form biofilms on a wide range of surfaces by synthesizing a cohesive and protective extracellular matrix. The morphology, internal structure and mechanical stability of a biofilm are largely determined by its constituent polymers. In addition to mediating adhesion to surfaces, biofilms control the uptake of molecules and regulate the permeability of the matrix to gases and chemicals. Since biofilms can cause significant problems in both industrial and healthcare settings, there is great interest in developing strategies that either inhibit their formation or facilitate their elimination. However, although important in this context, the material properties of bacterial biofilms are poorly understood. In particular, little is known about how the different components of a biofilm matrix contribute to its various physical characteristics, or how these are modified in response to environmental cues. In this review, we present an overview of the molecular composition of different bacterial biofilms and describe techniques for the characterization of their viscoelastic properties. Finally, we summarize our current understanding of how the mechanical properties of bacterial biofilms are altered by different environmental challenges, and we discuss initial insights into the relationship between these responses and the composition of the matrix.

Surface topology affects wetting behavior of Bacillus subtilis biofilms.

The colonization of surfaces by bacterial biofilms constitutes a huge problem in healthcare and industry. When attempting biofilm inactivation or removal, it is crucial to sufficiently wet the biofilm surface with antibacterial agents; however, certain biofilms efficiently resist wetting, and the origin of this behavior remains to date unclear. Here, we demonstrate that, depending on the growth medium used, the model bacterium Bacillus subtilis can form biofilm colonies with distinct surface properties: we find either hydrophilic or two variants of hydrophobic behavior. We show that those differences in biofilm wetting correlate with distinct surface topologies which, in turn, give rise to different physical wetting regimes known from lotus leaves or rose petals. Forming biofilms with different wetting properties may help bacteria to survive in both arid and humid conditions. Furthermore, converting the surface polarity of a biofilm could facilitate their removal from surfaces by increasing their wettability.

Amount of colicin release in Escherichia coli is regulated by lysis gene expression of the colicin E2 operon.

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.

Chemical warfare and survival strategies in bacterial range expansions.

Dispersal of species is a fundamental ecological process in the evolution and maintenance of biodiversity. Limited control over ecological parameters has hindered progress in understanding of what enables species to colonize new areas, as well as the importance of interspecies interactions. Such control is necessary to construct reliable mathematical models of ecosystems. In our work, we studied dispersal in the context of bacterial range expansions and identified the major determinants of species coexistence for a bacterial model system of three Escherichia coli strains (toxin-producing, sensitive and resistant). Genetic engineering allowed us to tune strain growth rates and to design different ecological scenarios (cyclic and hierarchical). We found that coexistence of all strains depended on three strongly interdependent factors: composition of inoculum, relative strain growth rates and effective toxin range. Robust agreement between our experiments and a thoroughly calibrated computational model enabled us to extrapolate these intricate interdependencies in terms of phenomenological biodiversity laws. Our mathematical analysis also suggested that cyclic dominance between strains is not a prerequisite for coexistence in competitive range expansions. Instead, robust three-strain coexistence required a balance between growth rates and either a reduced initial ratio of the toxin-producing strain, or a sufficiently short toxin range.

Heavy water reduces GFP expression in prokaryotic cell-free assays at the translation level while stimulating its transcription.

The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.