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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.

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., FEMS Microbiology Reviews, 45 (6), ISSN:0168-6445, ISSN:1574-6976

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.

Summary Temperature is one of the key cues that enable microorganisms to adjust their physiology in response to environmental changes. Here we show that motility is the major cellular function of Escherichia coli that is differentially regulated between growth at normal host temperature of 37°C and the febrile temperature of 42°C. Expression of both class II and class III flagellar genes is reduced at 42°C because of lowered level of the upstream activator FlhD. Class III genes are additionally repressed because of the destabilization and malfunction of secretion apparatus at high temperature, which prevents secretion of the anti-sigma factor FlgM. This mechanism of repression apparently accelerates loss of motility at 42°C. We hypothesize that E. coli perceives high temperature as a sign of inflammation, downregulating flagella to escape detection by the immune system of the host. Secretion-dependent coupling of gene expression to the environmental temperature is likely common among many bacteria., Graphical Abstract, Highlights • E. coli motility is tightly turned off at febrile temperature (42°C) • Repression of motility is achieved at two levels of hierarchical gene regulation • Lowered FlhD level reduces expression of all flagellar genes • Impaired FlgM secretion tightens repression of class III genes, Microbiology; Microbial Physiology; Microbial Cell Structure; Molecular Microbiology

mBio, 12 (3), ISSN:2150-7511, ISSN:2161-2129

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., 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.

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., 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.

The microorganisms in the gastrointestinal (GI) tract can influence the metabolism, immunity, and behavior of animal hosts. Increasing evidence suggests that communication between the host and the microbiome also occurs in the opposite direction, with hormones and other host-secreted compounds being sensed by microorganisms. Here, we addressed one key aspect of the host–microbe communication by studying chemotaxis of a model commensal bacterium, Escherichia coli, to several compounds present abundantly in the GI tract, namely catecholamines, thyroid hormones, and polyamines. Our results show that E. coli reacts to five out of ten analyzed chemicals, sensing melatonin, and spermidine as chemorepellents and showing mixed responses to dopamine, norepinephrine and 3,4-dihydroxymandelic acid. The strongest repellent response was observed for the polyamine spermidine, and we demonstrate that this response involves the low-abundance chemoreceptor Trg and the periplasmic binding protein PotD of the spermidine uptake system. The chemotactic effects of the tested compounds apparently correlate with their influence on growth and their stability in the GI tract, pointing to the specificity of the observed behavior. We hypothesize that the repellent responses observed at high concentrations of chemoeffective compounds might enable bacteria to avoid harmful levels of hormones and polyamines in the gut and, more generally, antimicrobial activities of the mucous layer.

Significance Whether residing in or invading the host, enterobacteria have to deal with host-related stress conditions. These stress factors also serve as sensory cues, informing bacteria that they are present inside the host. Here, we report that the PhoQ/PhoP two-component system, which was known to sense several host-related environmental changes, responds to osmotic upshift, another key stimulus associated with the host. This sensing is proposed to rely on a mechanism that detects changes in the physical properties of the membrane. Thus, a single enterobacterial kinase, PhoQ, senses a major part of host-associated stimuli. The PhoQ-mediated osmosensing increases bacterial fitness under hyperosmotic conditions found inside the host, and it is likely to play an important role in the regulation of virulence., 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.

Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems. Cells rely on their proteins being positioned correctly for processes such as cell division and migration. A model based on Turing patterns provides an active mechanism for establishing this precise control in bacteria.

The dynamics of microbial communities are heavily shaped by bacterium-bacteriophage interactions. But despite the apparent importance of bacteriophages, our understanding of the mechanisms controlling phage dynamics in bacterial populations, and particularly of the differences between the decisions that are made in the dormant lysogenic and active lytic states, remains limited. In this report, we show that enterobacterial phage T1, previously described as a lytic phage, is able to undergo lysogeny. We further demonstrate that the lysogeny-to-lysis decision occurs in response to changes in the density of the bacterial population, mediated by interspecies quorum-sensing signal AI-2, and in the metabolic state of the cell, mediated by cAMP receptor protein. We hypothesize that this strategy enables the phage to maximize its chances of self-amplification and spreading in bacterial population upon induction of the lytic cycle and that it might be common in phage-host interactions., Bacterial viruses, or bacteriophages, are highly abundant in the biosphere and have a major impact on microbial populations. Many examples of phage interactions with their hosts, including establishment of dormant lysogenic and active lytic states, have been characterized at the level of the individual cell. However, much less is known about the dependence of these interactions on host metabolism and signal exchange within bacterial communities. In this report, we describe a lysogenic state of the enterobacterial phage T1, previously known as a classical lytic phage, and characterize the underlying regulatory circuitry. We show that the transition from lysogeny to lysis depends on bacterial population density, perceived via interspecies autoinducer 2. Lysis is further controlled by the metabolic state of the cell, mediated by the cyclic-3′,5′-AMP (cAMP) receptor protein (CRP) of the host. We hypothesize that such combinations of cell density and metabolic sensing may be common in phage-host interactions.

Temperature is one of the key cues that enable microorganisms to adjust their physiology in response to environmental changes. Here we show that motility is the major cellular function of Escherichia coli that is differentially regulated between growth at normal host temperature of 37°C and the febrile temperature of 42°C. We demonstrate that this downregulation primarily occurs due to the malfunction of secretion apparatus, apparently related to the disassembly of the flagellar basal body. This prevented secretion of the anti-sigma factor FlgM and resulted in FlgM-dependent repression of class III motility genes at 42°C, while enabling prompt reactivation of motility upon decrease of temperature. We hypothesize that E. coli perceives high temperature as a sign of inflammation, downregulating flagella to escape detection by immune system of the host. Secretion-dependent coupling of gene expression to the environmental temperature represents a novel mode of stress response, which is likely to be common among many bacterial species.

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.

Most bacteria use transmembrane sensors to detect a wide range of environmental stimuli. A large class of such sensors are the chemotaxis receptors used by motile bacteria to follow environmental chemical gradients. In Escherichia coli, chemotaxis receptors are known to mediate highly sensitive responses to ligands, making them potentially useful for biosensory applications. However, with only four ligand-binding chemotaxis receptors, the natural ligand spectrum of E. coli is limited. The design of novel chemoreceptors to extend the sensing capabilities of E. coli is therefore a critical aspect of chemotaxis-based biosensor development. One path for novel sensor design is to harvest the large natural diversity of chemosensory functions found in bacteria by creating hybrids that have the signaling domain from E. coli chemotaxis receptors and sensory domains from other species. In this work, we demonstrate that the E. coli receptor Tar can be successfully combined with most typical sensory domains found in chemotaxis receptors and in evolutionary-related two-component histidine kinases. We show that such functional hybrids can be generated using several different fusion points. Our work further illustrates how hybrid receptors could be used to quantitatively characterize ligand specificity of chemotaxis receptors and histidine kinases using standardized assays in E. coli.

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. We assessed 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 is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

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., Swimming bacteria perform collective motion at high cell density, yet it is unclear how this behaviour affects their ability to follow substance gradients in the environment. Here, Colin et al. address this question by studying motion of Escherichia coli in controlled chemical gradients.

Microorganisms use transmembrane sensory receptors to perceive a wide range of environmental factors. It is unclear how rapidly the sensory properties of these receptors can be modified when microorganisms adapt to novel environments. Here, we demonstrate experimentally that the response of an Escherichia coli chemotaxis receptor to its chemical ligands can be easily inverted by mutations at several sites along receptor sequence. We also perform molecular dynamics simulations to shed light on the mechanism of the transmembrane signaling by E. coli chemoreceptors. Finally, we use receptors with inverted signaling to map determinants that enable the same receptor to sense multiple environmental factors, including metal ions, aromatic compounds, osmotic pressure, and salt ions. Our findings demonstrate high plasticity of signaling and provide further insights into the mechanisms of stimulus sensing and processing by bacterial chemoreceptors., Bacteria use chemotaxis receptors to perceive environmental factors. Here, the authors show that mutations in a chemotaxis receptor can invert the sensory response, e.g. from attractant to repellent, and use these mutants to map regions that enable the receptor to sense multiple environmental factors.

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.

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.

Motile bacteria use chemotaxis to migrate towards environments that are favorable for growth and survival. The signaling pathway that mediates this behavior is largely conserved among prokaryotes, with Escherichia coli chemotaxis system being one of the simplest and the best studied. At the core of this pathway are the arrays of clustered chemoreceptors that detect, amplify and integrate various stimuli. Recent work provided deeper understanding of spatial organization and signal processing by these clusters and uncovered the variety of sensory mechanisms used to detect environmental stimuli. Moreover, studies of bacteria with different lifestyles have led to new insights into the diversity and evolutionary conservation of the chemotaxis pathway, as well as the physiological relevance of chemotactic behavior in different environments.

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. faecalis . IMPORTANCE 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.

Cellular networks are intrinsically subject to stochastic fluctuations, but analysis of the resulting noise remained largely limited to gene expression. The pathway controlling chemotaxis ofEscherichia coliprovides 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.

Biofilms are surface-associated structures formed by bacteria embedded in a self-produced matrix. Biofilms are exceptionally resistant to environmental stress, antimicrobial agents and host immune defense, and combating biofilms has recently become an important research topic. In this work, we present a biologically engineered system that can be applied against a wide range of biofilms formed by pathogenic and non-pathogenic bacteria. This system relies on Escherichia coli (disrupter) strain that was engineered to synthesize and secrete Dispersin B, an enzyme that can hydrolyze poly-N-acetyl glucosamine (PGA), a polymer found in the matrix of various bacterial biofilms. We show that the degradation of PGA by the disruptor strain results in the dispersion of the target biofilm. We propose that in the future this simple disrupter module can be combined with other biofilm detection and targeting systems aimed towards the destruction of an existing biofilm.

Summary In bacterial habitats, the ability to follow spatial gradients of environmental factors that affect growth and survival can be largely advantageous. The bacterial strategy for unidirectional chemotactic movement in gradients of typical attractants or repellents, such as nutrients or toxins, is well understood. Optimal levels of other factors, however, may be found at intermediate points of a gradient and thus require a bidirectional tactic movement towards the optimum. Here we investigate the chemotactic response of Escherichia coli to pH as an example of such bidirectional taxis. We confirm that E. coli uses chemotaxis to avoid both extremes of low and high pH and demonstrate that the sign of the response is inverted from base-seeking to acid-seeking at a well-defined value of pH. Such inversion is enabled by opposing pH sensing by the two major chemoreceptors, Tar and Tsr, such that the relative strength of the response is modulated by adaptive receptor methylation. We further demonstrate that the inversion point of the pH response can be adjusted in response to changes in the cell density.

Diverse families of molecular chaperones cooperate to effect protein homeostasis, but the extent and dynamics of direct interactions among chaperone systems within cells remain little studied. Here we used fluorescence resonance energy transfer to systematically map the network of pairwise interactions among the major Escherichia coli chaperones. We demonstrate that in most cases functional cooperation between chaperones within and across families involves physical complex formation, which pre-exists even in the absence of folding substrates. The observed connectivity of the overall chaperone network confirms its partitioning into sub-networks that are responsible for de novo protein folding and maturation and for refolding/disaggregation of misfolded proteins, respectively, and are linked by the Hsp70 system. We further followed heat-induced changes in the cellular chaperone network, revealing two distinct pathways that process heat-denatured substrates. Our data suggest that protein folding within cells relies on highly ordered and direct channelling of substrates between chaperone systems and provide a comprehensive view of the underlying interactions and of their dynamics.

Bacterial flagellar motor is a highly ordered and complex supramolecular structure that powers rotation of flagella and serves as a type III export apparatus for flagellar assembly. Motor biogenesis represents a formidable example of self-assembly, but little is known about early steps of the motor structure formation. Here we used a combination of fluorescence microscopy techniques to dissect the order of the motor assembly in Escherichia coli cells, to map in vivo the underlying protein interactions and to investigate dynamics of protein exchange in the assembled motor structure. Our data suggest that motor self-assembly is initiated by oligomerization of the membrane export apparatus protein FlhA, which is followed by the recruitment of the MS ring component FliF and by the ordered association of other motor proteins. The assembly process combines the hierarchy with cooperativity, whereby the association of each subsequent motor structure stabilizes the growing assembly. Our results provide a novel and so far the most complete view of the early steps in flagellar motor assembly and improve understanding of the motor structure and regulation.

The chemotaxis system of Escherichia coli is sensitive to small relative changes in ambient chemoattractant concentrations over a broad range. Interactions among receptors are crucial to this sensitivity, as is precise adaptation, the return of chemoreceptor activity to prestimulus levels in a constant chemoeffector environment through methylation and demethylation of receptors. Signal integration and cooperativity have been attributed to strongly coupled, mixed teams of receptors, but receptors become individually methylated according to their ligand occupancy states. Here, we present a model of dynamic signaling teams that reconciles strong coupling among receptors with receptor-specific methylation. Receptor trimers of dimers couple to form a honeycomb lattice, consistent with cryo-electron microscopy (cryoEM) tomography, within which the boundaries of signaling teams change rapidly. Our model helps explain the inferred increase in signaling team size with receptor modification, and indicates that active trimers couple more strongly than inactive trimers.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001, eLife digest A cell must duplicate its genetic material and then separate the two copies before it divides. This process is carefully controlled so that each new cell receives an identical set of chromosomes after cell division. In budding yeast, new daughter cells grow as a bud on the side a larger mother cell and are eventually pinched off. A surveillance mechanism in budding yeast monitors the placement of the molecular machine (called the spindle) that separates the copies of the chromosomes. This mechanism then stops the cell from dividing if the spindle is not positioned correctly. Many of the components of this surveillance mechanism – which is called the spindle position checkpoint – associate with structures at the ends of the spindle. However, it was not clear how these components do this and how it helps them to check if the spindle is positioned correctly. Now, Gryaznova, Caydasi et al. have used a technique called FRET to answer these questions for an important component of the spindle position checkpoint, a protein called Bfa1. The main advantage of FRET is that it can be used to monitor changes in protein-protein interactions in living cells. This approach identified two proteins that provide sites for Bfa1 to bind to at the ends of the spindle. The experiments also showed that Bfa1 specifically detaches from one of these proteins (called Spc72) when the spindle position checkpoint is activated. This action keeps Bfa1 (and therefore the spindle position checkpoint) active, which in turn stops the cell from starting to divide. Further experiments then showed that Spc72 acts like a regulatory hub that controls Bfa1’s activity. This allows an as-yet unidentified mechanism to coordinate cell division with the position of the spindle. The findings of Gryaznova, Caydasi et al. also suggest that unknown factors switch off the spindle position checkpoint when the spindle is correctly positioned to allow the cell to divide. Future work could now aim to identify the mechanism and the unknown factors. Finally, in a related study, Falk et al. show that the spindle position checkpoint is inactivated when one end of the spindle is moved out of the mother cell and into the bud. DOI: http://dx.doi.org/10.7554/eLife.14029.002

Summary 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, E scherichia coli and B acillus 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.

Taz is a chimeric receptor consisting of the periplasmic, transmembrane and most of the HAMP linker domains of the Escherichia coli aspartate receptor (Tar(Ec)) and the cytoplasmic signalling domain of the E. coli osmosensor EnvZ. Aspartate is one of several attractant ligands normally sensed by Tar and it interacts with Taz to induce OmpR-dependent transcription from the ompC promoter--albeit with reduced sensitivity relative to the chemotactic response it evokes via Tar. By combining Taz with a reporter system that expresses green fluorescent protein (GFP) from the ompC promoter, we were able to examine the interaction of Taz with all 20 natural amino acids. Some amino acids (Leu, Met, Val and Ser) reduced GFP expression, which in the case of leucine is likely attributed to a direct effect on the receptor, rather than an indirect effect through the leucine responsive protein (Lrp). Surprisingly, amino acids like Met and Ser--which are also attractants for Tar--'inhibited' Taz. Moreover, Taz exhibits a higher sensitivity to Leu compared with Asp, which is the inverse of Tar. Our results show the exquisite sensitivity of chemotactic receptors. Small conformational changes induced by making the chimera may have changed the way it responds to different amino acids.

Chemotaxis in Escherichia coli is one of the most thoroughly studied model systems for signal transduction. Receptor-kinase complexes, organized in clusters at the cell poles, sense chemoeffector stimuli and transmit signals to flagellar motors by phosphorylation of a diffusible response regulator protein. Despite the apparent simplicity of the signal transduction pathway, the high sensitivity, wide dynamic range and integration of multiple stimuli of this pathway remain unexplained. Recent advances in computer modeling and in quantitative experimental analysis suggest that cooperative protein interactions in receptor clusters play a crucial role in the signal processing during bacterial chemotaxis.

Bacterial chemoreceptors are embedded in the inner cell membrane in tight clusters. We show that changes in receptor methylation that generate large changes in kinase activity have relatively little effect on cluster morphology. Thus, changes in receptor activity do not appear to be mediated by changes in receptor-kinase assembly.

The existence and nature of an active chromosome segregation apparatus in bacteria has been a long-standing debate. A novel Brownian ratchet-type mechanism of chromosome segregation mediated by the Min system is identified in E. coli., Numerical simulations show that entropy alone is not sufficient to complete segregation of bacterial chromosomes. Chromosome segregation can be enhanced by a polar gradient of DNA tethering sites on the membrane. The cell-division regulator MinD forms a polar gradient on the membrane and binds DNA in an ATP-dependent manner. The bacterial Min system coordinates cell division and chromosome segregation., The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane-associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP-dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology.

Bacteria, fungi and plants rescue aggregated proteins using a powerful bichaperone system composed of an Hsp70 chaperone and an Hsp100 AAA+ disaggregase. In Escherichia coli, the Hsp70 chaperone DnaK binds aggregates and targets the disaggregase ClpB to the substrate. ClpB hexamers use ATP to thread substrate polypeptides through the central pore, driving disaggregation. How ClpB finds DnaK and regulates threading remains unclear. To dissect the disaggregation mechanism, we separated these steps using primarily chimeric ClpB-ClpV constructs that directly recognize alternative substrates, thereby obviating DnaK involvement. We show that ClpB has low intrinsic disaggregation activity that is normally repressed by the ClpB middle (M) domain. In the presence of aggregate, DnaK directly binds M-domain motif 2, increasing ClpB ATPase activity to unleash high ClpB threading power. Our results uncover a new function for Hsp70: the coupling of substrate targeting to AAA+ chaperone activation at aggregate surfaces.

Chemotaxis allows bacteria to follow gradients of nutrients and other environmental stimuli. The bacterium Escherichia coli performs chemotaxis via a run-and-tumble strategy in which sensitive temporal comparisons lead to a biased random walk, with longer runs in the preferred gradient direction. The chemotaxis network of E. coli has developed over the years into one of the most thoroughly studied model systems for signal transduction and behaviour, yielding general insights into such properties of cellular networks as signal amplification, signal integration, and robustness. Despite its relative simplicity, the operation of the E. coli chemotaxis network is highly refined and evolutionarily optimized at many levels. For example, recent studies revealed that the network adjusts its signaling properties dependent on the extracellular environment, apparently to optimize chemotaxis under particular conditions. The network can even utilize potentially detrimental stochastic fluctuations in protein levels and reaction rates to maximize the chemotactic performance of the population.

The chemotaxis network of the bacterium Escherichia coli is perhaps the most studied model for adaptation of a signaling system to persistent stimuli. Although adaptation in this system is generally considered to be precise, there has been little effort to quantify this precision, or to understand how and when precision fails. Using a Förster resonance energy transfer-based reporter of signaling activity, we undertook a systematic study of adaptation kinetics and precision in E. coli cells expressing a single type of chemoreceptor (Tar). Quantifiable loss of precision of adaptation was observed at levels of the attractant MeAsp as low 10 μM, with pronounced differences in both kinetics and precision of adaptation between addition and removal of attractant. Quantitative modeling of the kinetic data suggests that loss of precise adaptation is due to a slowing of receptor methylation as available modification sites become scarce. Moreover, the observed kinetics of adaptation imply large cell-to-cell variation in adaptation rates—potentially providing genetically identical cells with the ability to “hedge their bets” by pursuing distinct chemotactic strategies.

Adaptation of the chemotaxis sensory pathway of the bacterium Escherichia coli is integral for detecting chemicals over a wide range of background concentrations, ultimately allowing cells to swim towards sources of attractant and away from repellents. Its biochemical mechanism based on methylation and demethylation of chemoreceptors has long been known. Despite the importance of adaptation for cell memory and behavior, the dynamics of adaptation are difficult to reconcile with current models of precise adaptation. Here, we follow time courses of signaling in response to concentration step changes of attractant using in vivo fluorescence resonance energy transfer measurements. Specifically, we use a condensed representation of adaptation time courses for efficient evaluation of different adaptation models. To quantitatively explain the data, we finally develop a dynamic model for signaling and adaptation based on the attractant flow in the experiment, signaling by cooperative receptor complexes, and multiple layers of feedback regulation for adaptation. We experimentally confirm the predicted effects of changing the enzyme-expression level and bypassing the negative feedback for demethylation. Our data analysis suggests significant imprecision in adaptation for large additions. Furthermore, our model predicts highly regulated, ultrafast adaptation in response to removal of attractant, which may be useful for fast reorientation of the cell and noise reduction in adaptation., Comment: accepted for publication in PLoS Computational Biology; manuscript (19 pages, 5 figures) and supplementary information; added additional clarification on alternative adaptation models in supplementary information

Escherichia coli chemotaxis has long served as a simple model of environmental signal processing, and bacterial responses to single chemical gradients are relatively well understood. Less is known about the chemotactic behavior of E. coli in multiple chemical gradients. In their native environment, cells are often exposed to multiple chemical stimuli. Using a recently developed microfluidic chemotaxis device, we exposed E. coli cells to two opposing but equally potent gradients of major attractants, methyl-aspartate and serine. The responses of E. coli cells demonstrated that chemotactic decisions depended on the ratio of the respective receptor number of Tar/Tsr. In addition, the ratio of Tar to Tsr was found to vary with cells’ growth conditions, whereby it depended on the culture density but not on the growth duration. These results provide biological insights into the decision-making processes of chemotactic bacteria that are subjected to multiple chemical stimuli and demonstrate the importance of the cellular microenvironment in determining phenotypic behavior.

Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.