Thermal robustness of signaling in bacterial chemotaxis.

Temperature is a global factor that affects the performance of all intracellular networks. Robustness against temperature variations is thus expected to be an essential network property, particularly in organisms without inherent temperature control. Here, we combine experimental analyses with computational modeling to investigate thermal robustness of signaling in chemotaxis of Escherichia coli, a relatively simple and well-established model for systems biology. We show that steady-state and kinetic pathway parameters that are essential for chemotactic performance are indeed temperature-compensated in the entire physiological range. Thermal robustness of steady-state pathway output is ensured at several levels by mutual compensation of temperature effects on activities of individual pathway components. Moreover, the effect of temperature on adaptation kinetics is counterbalanced by preprogrammed temperature dependence of enzyme synthesis and stability to achieve nearly optimal performance at the growth temperature. Similar compensatory mechanisms are expected to ensure thermal robustness in other systems.

Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli.

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Second messenger-mediated adjustment of bacterial swimming velocity.

Bacteria swim by means of rotating flagella that are powered by ion influx through membrane-spanning motor complexes. Escherichia coli and related species harness a chemosensory and signal transduction machinery that governs the direction of flagellar rotation and allows them to navigate in chemical gradients. Here, we show that Escherichia coli can also fine-tune its swimming speed with the help of a molecular brake (YcgR) that, upon binding of the nucleotide second messenger cyclic di-GMP, interacts with the motor protein MotA to curb flagellar motor output. Swimming velocity is controlled by the synergistic action of at least five signaling proteins that adjust the cellular concentration of cyclic di-GMP. Activation of this network and the resulting deceleration coincide with nutrient depletion and might represent an adaptation to starvation. These experiments demonstrate that bacteria can modulate flagellar motor output and thus swimming velocity in response to environmental cues.

Growth-rate dependent resource investment in bacterial motile behavior quantitatively follows potential benefit of chemotaxis.

Microorganisms possess diverse mechanisms to regulate investment into individual cellular processes according to their environment. How these regulatory strategies reflect the inherent trade-off between the benefit and cost of resource investment remains largely unknown, particularly for many cellular functions that are not immediately related to growth. Here, we investigate regulation of motility and chemotaxis, one of the most complex and costly bacterial behaviors, as a function of bacterial growth rate. We show with experiment and theory that in poor nutritional conditions, Escherichia coli increases its investment in motility in proportion to the reproductive fitness advantage provided by the ability to follow nutrient gradients. Since this growth-rate dependent regulation of motility genes occurs even when nutrient gradients are absent, we hypothesize that it reflects an anticipatory preallocation of cellular resources. Notably, relative fitness benefit of chemotaxis could be observed not only in the presence of imposed gradients of secondary nutrients but also in initially homogeneous bacterial cultures, suggesting that bacteria can generate local gradients of carbon sources and excreted metabolites, and subsequently use chemotaxis to enhance the utilization of these compounds. This interplay between metabolite excretion and their chemotaxis-dependent reutilization is likely to play an important general role in microbial communities.

Chemotaxis and cyclic-di-GMP signalling control surface attachment of Escherichia coli.

Attachment to surfaces is an important early step during bacterial infection and during formation of submerged biofilms. Although flagella-mediated motility is known to be important for attachment of Escherichia coli and other bacteria, implications of motility regulation by cellular signalling remain to be understood. Here, we show that motility largely promotes attachment of E. coli, including that mediated by type 1 fimbriae, by allowing cells to reach, get hydrodynamically trapped at and explore the surface. Inactivation or inhibition of the chemotaxis signalling pathway improves attachment by suppressing cell reorientations and thereby increasing surface residence times. The attachment is further enhanced by deletion of genes encoding the cyclic diguanosine monophosphate (c-di-GMP)-dependent flagellar brake YcgR or the diguanylate cyclase DgcE. Such increased attachment in absence of c-di-GMP signalling is in contrast to its commonly accepted function as a positive regulator of the sessile state. It is apparently due to the increased swimming speed of E. coli in absence of YcgR-mediated motor control, which strengthens adhesion mediated by the type 1 fimbriae. Thus, both signalling networks that regulate motility of E. coli also control its engagement with both biotic and abiotic surfaces, which has likely implications for infection and biofilm formation.

The protein architecture of the endocytic coat analyzed by FRET microscopy.

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here, we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below the lattice. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the coat. We propose that this design enables an efficient and regulated function of the endocytic coat during endocytic vesicle formation.

Functional determinants of a small protein controlling a broadly conserved bacterial sensor kinase.

The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella and related bacteria. Previous studies have shown that MgrB, a 47 amino acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine scanning mutagenesis of the periplasmic region of MgrB further indicates that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms.

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface.IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

Osmosensing by the bacterial PhoQ/PhoP two-component system.

The PhoQ/PhoP two-component system plays an essential role in the response of enterobacteria to the environment of their mammalian hosts. It is known to sense several stimuli that are potentially associated with the host, including extracellular magnesium limitation, low pH, and the presence of cationic antimicrobial peptides. Here, we show that the PhoQ/PhoP two-component systems of Escherichia coli and Salmonella can also perceive an osmotic upshift, another key stimulus to which bacteria become exposed within the host. In contrast to most previously established stimuli of PhoQ, the detection of osmotic upshift does not require its periplasmic sensor domain. Instead, we show that the activity of PhoQ is affected by the length of the transmembrane (TM) helix as well as by membrane lateral pressure. We therefore propose that osmosensing relies on a conformational change within the TM domain of PhoQ induced by a perturbation in cell membrane thickness and lateral pressure under hyperosmotic conditions. Furthermore, the response mediated by the PhoQ/PhoP two-component system was found to improve bacterial growth recovery under hyperosmotic stress, partly through stabilization of the sigma factor RpoS. Our findings directly link the PhoQ/PhoP two-component system to bacterial osmosensing, suggesting that this system can mediate a concerted response to most of the established host-related cues.

Transmembrane region of bacterial chemoreceptor is capable of promoting protein clustering.

Many membrane proteins are known to form higher-order oligomers, but the degree to which membrane regions could facilitate protein complex assembly remains largely unclear. Clusters of chemotaxis receptors are among the most prominent structures in the bacterial cell membrane, and they play important functions in processing of chemotactic signals. Although much work has been done to elucidate mechanisms of cluster formation, it almost exclusively focused on cytoplasmic interactions among receptors and other chemotaxis proteins, whereas involvement of membrane-mediated interactions was only hypothesized. Here we used imaging of constructs composed of only a fluorescent protein and the TM helices of Tar to demonstrate that interactions between the lipid bilayer and transmembrane (TM) helices of Escherichia coli chemoreceptors alone are sufficient to mediate clustering. We found that the ability to cluster depends on the sequence or length of the TM helices, implying that certain conformations of these helices facilitate clustering, whereas others do not. Notably, observed sequence specificity was apparently consistent with differences in clustering between native E. coli receptors, with the TM sequence of better-clustering high-abundance receptors being more efficient in promoting membrane-mediated complex formation. These results indicate that being more than just membrane anchors, TM helices could play an important role in the clustering and organization of membrane proteins in bacteria.

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System.

A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid-derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP-regenerating and oxygen-scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom-up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.

Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.

The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy.

Sharing of Phosphatases Promotes Response Plasticity in Phosphorylation Cascades.

Sharing of positive or negative regulators between multiple targets is frequently observed in cellular signaling cascades. For instance, phosphatase sharing between multiple kinases is ubiquitous within the MAPK pathway. Here we investigate how such phosphatase sharing could shape robustness and evolvability of the phosphorylation cascade. Through modeling and evolutionary simulations, we demonstrate that 1) phosphatase sharing dramatically increases robustness of a bistable MAPK response, and 2) phosphatase-sharing cascades evolve faster than nonsharing cascades. This faster evolution is particularly pronounced when evolving from a monostable toward a bistable phenotype, whereas the transition speed of a population from a bistable to monostable response is not affected by phosphatase sharing. This property may enable the phosphatase-sharing design to adapt better in a changing environment. Analysis of the respective mutational landscapes reveal that phosphatase sharing reduces the number of limiting mutations required for transition from monostable to bistable responses, hence facilitating a faster transition to such response types. Taken together, using MAPK cascade as an example, our study offers a general theoretical framework to explore robustness and evolutionary plasticity of signal transduction cascades.

Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture.

Biofilms in nature typically consist of multiple species, and microbial interactions are likely to have crucial effects on biofilm development, structure, and functions. The best-understood form of communication within bacterial communities involves the production, release, and detection of signal molecules (autoinducers), known as quorum sensing. Although autoinducers mainly promote intraspecies communication, autoinducer 2 (AI-2) is produced and detected by a variety of bacteria, thus principally allowing interspecies communication. Here we show the importance of AI-2-mediated signaling in the formation of mixed biofilms by Enterococcus faecalis and Escherichia coli Our results demonstrate that AI-2 produced by E. faecalis promotes collective behaviors of E. coli at lower cell densities, enhancing autoaggregation of E. coli but also leading to chemotaxis-dependent coaggregation between the two species. Finally, we show that formation of such mixed dual-species biofilms increases the stress resistance of both E. coli and E. faecalisIMPORTANCE The role of interspecies communication in the development of mixed microbial communities is becoming increasingly apparent, but specific examples of such communication remain limited. The universal signal molecule AI-2 is well known to regulate cell-density-dependent phenotypes of many bacterial species but, despite its potential for interspecies communication, the role of AI-2 in the establishment of multispecies communities is not well understood. In this study, we explore AI-2 signaling in a dual-species community containing two bacterial species that naturally cooccur in their mammalian hosts, i.e., Escherichia coli and Enterococcus faecalis We show that active production of AI-2 by E. faecalis allows E. coli to perform collective behaviors at low cell densities. Additionally, AI-2- and chemotaxis-dependent coaggregation with E. faecalis creates nucleation zones for rapid growth of E. coli microcolonies in mixed biofilms and enhances the stress resistance of both species.

MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Drift and Behavior of E. coli Cells.

Chemotaxis of the bacterium Escherichia coli is well understood in shallow chemical gradients, but its swimming behavior remains difficult to interpret in steep gradients. By focusing on single-cell trajectories from simulations, we investigated the dependence of the chemotactic drift velocity on attractant concentration in an exponential gradient. Whereas maxima of the average drift velocity can be interpreted within analytical linear-response theory of chemotaxis in shallow gradients, limits in drift due to steep gradients and finite number of receptor-methylation sites for adaptation go beyond perturbation theory. For instance, we found a surprising pinning of the cells to the concentration in the gradient at which cells run out of methylation sites. To validate the positions of maximal drift, we recorded single-cell trajectories in carefully designed chemical gradients using microfluidics.

Secondary bacterial flagellar system improves bacterial spreading by increasing the directional persistence of swimming.

As numerous bacterial species, Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system. A significant subpopulation of CN-32 cells induces expression of the secondary system under planktonic conditions, resulting in formation of one, sometimes two, filaments at lateral positions in addition to the primary polar flagellum. Mutant analysis revealed that the single chemotaxis system primarily or even exclusively addresses the main polar flagellar system. Cells with secondary filaments outperformed their monopolarly flagellated counterparts in spreading on soft-agar plates and through medium-filled channels despite having lower swimming speed. While mutant cells with only polar flagella navigate by a "run-reverse-flick" mechanism resulting in effective cell realignments of about 90°, wild-type cells with secondary filaments exhibited a range of realignment angles with an average value of smaller than 90°. Mathematical modeling and computer simulations demonstrated that the smaller realignment angle of wild-type cells results in the higher directional persistence, increasing spreading efficiency both with and without a chemical gradient. Taken together, we propose that in S. putrefaciens CN-32, cell propulsion and directional switches are mainly mediated by the polar flagellar system, while the secondary filament increases the directional persistence of swimming and thus of spreading in the environment.

Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa.

The PctA and PctB chemoreceptors of Pseudomonas aeruginosa mediate chemotaxis toward amino acids. A general feature of signal transduction processes is that a signal input is converted into an output. We have generated chimeras combining the Tar signaling domain with either the PctA or PctB ligand binding domain (LBD). Escherichia coli harboring either PctA-Tar or PctB-Tar mediated chemotaxis toward amino acids. The responses of both chimeras were determined using fluorescence resonance energy transfer, and the derived EC50 values are a measure of output. PctA-Tar and PctB-Tar responded to 19 and 11 L-amino acids respectively. The EC50 values of PctA-Tar responses differed by more than three orders of magnitude, whereas PctB-Tar responded preferentially to L-Gln. The comparison of amino acid binding constants and the corresponding EC50 values for both receptors revealed statistically significant correlations between inputs and outputs. PctA and PctB possess a double PDC (PhoQ-DcuS-CitA) LBD - a family of binding domain found in various other amino acid chemoreceptors. Similarly, various chemoreceptors share the preferential response to certain amino acids (e.g. L-Cys, L-Ser and L-Thr) that we observed for PctA. Defining the specific inputs and outputs of these chemoreceptors is an important step toward better understanding of their physiological role.

Relation between chemotaxis and consumption of amino acids in bacteria.

Chemotaxis enables bacteria to navigate chemical gradients in their environment, accumulating toward high concentrations of attractants and avoiding high concentrations of repellents. Although finding nutrients is likely to be an important function of bacterial chemotaxis, not all characterized attractants are nutrients. Moreover, even for potential nutrients, the exact relation between the metabolic value of chemicals and their efficiency as chemoattractants has not been systematically explored. Here we compare the chemotactic response of amino acids with their use by bacteria for two well-established models of chemotactic behavior, Escherichia coli and Bacillus subtilis. We demonstrate that in E. coli chemotaxis toward amino acids indeed strongly correlates with their utilization. However, no such correlation is observed for B. subtilis, suggesting that in this case, the amino acids are not followed because of their nutritional value but rather as environmental cues.