D-amino acids signal a stress-dependent run-away response in Vibrio cholerae.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release D-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that D-arginine and D-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These D-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either D-arginine or D-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these D-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that D-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Dynamic fluctuations in a bacterial metabolic network.

The operation of the central metabolism is typically assumed to be deterministic, but dynamics and high connectivity of the metabolic network make it potentially prone to generating fluctuations. However, time-resolved measurements of metabolite levels in individual cells that are required to characterize such fluctuations remained a challenge, particularly in small bacterial cells. Here we use single-cell metabolite measurements based on Förster resonance energy transfer, combined with computer simulations, to explore the real-time dynamics of the metabolic network of Escherichia coli. We observe that steplike exposure of starved E. coli to glycolytic carbon sources elicits large periodic fluctuations in the intracellular concentration of pyruvate in individual cells. These fluctuations are consistent with predicted oscillatory dynamics of E. coli metabolic network, and they are primarily controlled by biochemical reactions around the pyruvate node. Our results further indicate that fluctuations in glycolysis propagate to other cellular processes, possibly leading to temporal heterogeneity of cellular states within a population.

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth.

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here, we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

Running after your host: Stimulation of bacterial motility promotes colonization.

In this issue of Cell Host and Microbe, Robinson et al. (2021) make elegant use of experimental evolution to demonstrate that increased motility promotes migration toward and colonization of zebrafish larvae by a commensal bacterium. Stimulation of motility depends on bacterial second messenger and on signals released by resident host microbiota.

Multiple functions of flagellar motility and chemotaxis in bacterial physiology.

Most swimming bacteria are capable of following gradients of nutrients, signaling molecules and other environmental factors that affect bacterial physiology. This tactic behavior became one of the most-studied model systems for signal transduction and quantitative biology, and underlying molecular mechanisms are well characterized in Escherichia coli and several other model bacteria. In this review, we focus primarily on less understood aspect of bacterial chemotaxis, namely its physiological relevance for individual bacterial cells and for bacterial populations. As evident from multiple recent studies, even for the same bacterial species flagellar motility and chemotaxis might serve multiple roles, depending on the physiological and environmental conditions. Among these, finding sources of nutrients and more generally locating niches that are optimal for growth appear to be one of the major functions of bacterial chemotaxis, which could explain many chemoeffector preferences as well as flagellar gene regulation. Chemotaxis might also generally enhance efficiency of environmental colonization by motile bacteria, which involves intricate interplay between individual and collective behaviors and trade-offs between growth and motility. Finally, motility and chemotaxis play multiple roles in collective behaviors of bacteria including swarming, biofilm formation and autoaggregation, as well as in their interactions with animal and plant hosts.

Production and Characterization of Motile and Chemotactic Bacterial Minicells.

Minicells are nanosized membrane vesicles produced by bacteria. Minicells are chromosome-free but contain cellular biosynthetic and metabolic machinery, and they are robust due to the protection provided by the bacterial cell envelope, which makes them potentially highly attractive in biomedical applications. However, the applicability of minicells and other nanoparticle-based delivery systems is limited by their inefficient accumulation at the target. Here we engineered the minicell-producing Escherichia coli strain to overexpress flagellar genes, which enables the generation of motile minicells. We subsequently performed an experimental and theoretical analysis of the minicell motility and their responses to gradients of chemoeffectors. Despite important differences between the motility of minicells and normal bacterial cells, minicells were able to bias their movement in chemical gradients and to accumulate toward the sources of chemoattractants. Such motile and chemotactic minicells may thus be applicable for an active effector delivery and specific targeting of tissues and cells according to their metabolic profiles.

Asymmetric mating behavior of isogamous budding yeast.

Anisogamy, the size difference between small male and large female gametes, is known to enable selection for sexual dimorphism and behavioral differences between sexes. Nevertheless, even isogamous species exhibit molecular asymmetries between mating types, which are known to ensure their self-incompatibility. Here, we show that different properties of the pheromones secreted by the MATa and MATα mating types of budding yeast lead to asymmetry in their behavioral responses during mating in mixed haploid populations, which resemble behavioral asymmetries between gametes in anisogamous organisms. MATa behaves as a random searcher that is stimulated in proportion to the fraction of MATα partner cells within the population, whereas MATα behaves as a short-range directional distance sensor. Mathematical modeling suggests that the observed asymmetric responses can enhance efficiency of mating and might thus provide a selective advantage. Our results demonstrate that the emergence of asymmetric mating behavior did not require anisogamy-based sexual selection.

Flagellum-Mediated Mechanosensing and RflP Control Motility State of Pathogenic Escherichia coli.

Bacterial flagellar motility plays an important role in many processes that occur at surfaces or in hydrogels, including adhesion, biofilm formation, and bacterium-host interactions. Consequently, expression of flagellar genes, as well as genes involved in biofilm formation and virulence, can be regulated by the surface contact. In a few bacterial species, flagella themselves are known to serve as mechanosensors, where an increased load on flagella experienced during surface contact or swimming in viscous media controls gene expression. In this study, we show that gene regulation by motility-dependent mechanosensing is common among pathogenic Escherichia coli strains. This regulatory mechanism requires flagellar rotation, and it enables pathogenic E. coli to repress flagellar genes at low loads in liquid culture, while activating motility in porous medium (soft agar) or upon surface contact. It also controls several other cellular functions, including metabolism and signaling. The mechanosensing response in pathogenic E. coli depends on the negative regulator of motility, RflP (YdiV), which inhibits basal expression of flagellar genes in liquid. While no conditional inhibition of flagellar gene expression in liquid and therefore no upregulation in porous medium was observed in the wild-type commensal or laboratory strains of E. coli, mechanosensitive regulation could be recovered by overexpression of RflP in the laboratory strain. We hypothesize that this conditional activation of flagellar genes in pathogenic E. coli reflects adaptation to the dual role played by flagella and motility during infection.IMPORTANCE Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.

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.

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.

Chemotactic behaviour of Escherichia coli at high cell density.

At high cell density, swimming bacteria exhibit collective motility patterns, self-organized through physical interactions of a however still debated nature. Although high-density behaviours are frequent in natural situations, it remained unknown how collective motion affects chemotaxis, the main physiological function of motility, which enables bacteria to follow environmental gradients in their habitats. Here, we systematically investigate this question in the model organism Escherichia coli, varying cell density, cell length, and suspension confinement. The characteristics of the collective motion indicate that hydrodynamic interactions between swimmers made the primary contribution to its emergence. We observe that the chemotactic drift is moderately enhanced at intermediate cell densities, peaks, and is then strongly suppressed at higher densities. Numerical simulations reveal that this suppression occurs because the collective motion disturbs the choreography necessary for chemotactic sensing. We suggest that this physical hindrance imposes a fundamental constraint on high-density behaviours of motile bacteria, including swarming and the formation of multicellular aggregates and biofilms.

Motility and chemotaxis of bacteria-driven microswimmers fabricated using antigen 43-mediated biotin display.

Bacteria-driven biohybrid microswimmers (bacteriabots) combine synthetic cargo with motile living bacteria that enable propulsion and steering. Although fabrication and potential use of such bacteriabots have attracted much attention, existing methods of fabrication require an extensive sample preparation that can drastically decrease the viability and motility of bacteria. Moreover, chemotactic behavior of bacteriabots in a liquid medium with chemical gradients has remained largely unclear. To overcome these shortcomings, we designed Escherichia coli to autonomously display biotin on its cell surface via the engineered autotransporter antigen 43 and thus to bind streptavidin-coated cargo. We show that the cargo attachment to these bacteria is greatly enhanced by motility and occurs predominantly at the cell poles, which is greatly beneficial for the fabrication of motile bacteriabots. We further performed a systemic study to understand and optimize the ability of these bacteriabots to follow chemical gradients. We demonstrate that the chemotaxis of bacteriabots is primarily limited by the cargo-dependent reduction of swimming speed and show that the fabrication of bacteriabots using elongated E. coli cells can be used to overcome this limitation.

Activity statistics in a colloidal glass former: Experimental evidence for a dynamical transition.

In a dense colloidal suspension at a volume fraction below the glass transition, we follow the trajectories of an assembly of tracers over a large time window. We define a local activity, which quantifies the local tendency of the system to rearrange. We determine the statistics of the time integrated activity, and we argue that it develops a low activity tail that comes together with the onset of glassy-like behavior and heterogeneous dynamics. These rare events may be interpreted as the reflection of an underlying dynamic phase transition.

Multiple sources of slow activity fluctuations in a bacterial chemosensory network.

Cellular networks are intrinsically subject to stochastic fluctuations, but analysis of the resulting noise remained largely limited to gene expression. The pathway controlling chemotaxis of Escherichia coli provides one example where posttranslational signaling noise has been deduced from cellular behavior. This noise was proposed to result from stochasticity in chemoreceptor methylation, and it is believed to enhance environment exploration by bacteria. Here we combined single-cell FRET measurements with analysis based on the fluctuation-dissipation theorem (FDT) to characterize origins of activity fluctuations within the chemotaxis pathway. We observed surprisingly large methylation-independent thermal fluctuations of receptor activity, which contribute to noise comparably to the energy-consuming methylation dynamics. Interactions between clustered receptors involved in amplification of chemotactic signals are also necessary to produce the observed large activity fluctuations. Our work thus shows that the high response sensitivity of this cellular pathway also increases its susceptibility to noise, from thermal and out-of-equilibrium processes.

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.

Emergent properties of bacterial chemotaxis pathway.

The chemotaxis pathway of Escherichia coli is the most studied sensory system in prokaryotes. The highly conserved general architecture of this pathway consists of two modules which mediate signal transduction and adaptation. The signal transduction module detects and amplifies changes in environmental conditions and rapidly transmits these signals to control bacterial swimming behavior. The adaptation module gradually resets the activity and sensitivity of the first module after initial stimulation and thereby enables the temporal comparisons necessary for bacterial chemotaxis. Recent experimental and theoretical work has unraveled multiple quantitative features emerging from the interplay between these two modules. This has laid the groundwork for rationalization of these emerging properties in the context of the evolutionary optimization of the chemotactic behavior.

Evolutionary Remodeling of Bacterial Motility Checkpoint Control.

Regulatory networks play a central role in the relationship between genotype and phenotype in all organisms. However, the mechanisms that underpin the evolutionary plasticity of these networks remain poorly understood. Here, we used experimental selection for enhanced bacterial motility in a porous environment to explore the adaptability of one of the most complex networks known in bacteria. We found that the resulting phenotypic changes are mediated by adaptive mutations in several functionally different proteins, including multiple components of the flagellar motor. Nevertheless, this evolutionary adaptation could be explained by a single mechanism, namely remodeling of the checkpoint regulating flagellar gene expression. Supported by computer simulations, our findings suggest that the specific "bow-tie" topology of the checkpoint facilitates evolutionary tuning of the cost-benefit trade-off between motility and growth. We propose that bow-tie regulatory motifs, which are widespread in cellular networks, play a general role in evolutionary adaptation.

Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli.

Bacteria communicate by producing and sensing extracellular signal molecules called autoinducers. Such intercellular signalling, known as quorum sensing, allows bacteria to coordinate and synchronize behavioural responses at high cell densities. Autoinducer 2 (AI-2) is the only known quorum-sensing molecule produced by Escherichia coli but its physiological role remains elusive, although it is known to regulate biofilm formation and virulence in other bacterial species. Here we show that chemotaxis towards self-produced AI-2 can mediate collective behaviour-autoaggregation-of E. coli. Autoaggregation requires motility and is strongly enhanced by chemotaxis to AI-2 at physiological cell densities. These effects are observed regardless whether cell-cell interactions under particular growth conditions are mediated by the major E. coli adhesin (antigen 43) or by curli fibres. Furthermore, AI-2-dependent autoaggregation enhances bacterial stress resistance and promotes biofilm formation.