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Key Points Chemotaxis allows bacteria to swim towards environments that are better for growth. The process is involved in pathogenicity, biofilm formation and the establishment of symbiotic relationships. Changes in attractant and repellent concentrations are detected by clusters of chemoreceptors. Bacteria can sense very small changes in attractant concentration over a wide range of background concentrations. The chemoreceptor clusters control the activity of a two-component system comprising the histidine protein kinase CheA and the response regulators CheY and CheB. Phosphorylated CheY controls flagellar motor switching, whereas phosphorylated CheB mediates adaptation. The Escherichia coli chemotaxis signalling pathway is one of the simplest and best understood, but it is becoming increasingly apparent that most bacteria have more complex chemosensory pathways involving multiple homologues of the E. coli chemotaxis proteins. Rhodobacter sphaeroides has one of the best understood complex chemotaxis pathways; it has two distinct types of chemosensory cluster: one that is positioned at the cell pole and detects changes in the external attractant and repellent concentrations, and another that is cytoplasmic and is believed to monitor the metabolic state of the cell (a form of energy taxis). Structural studies have revealed the specificity determinants in the interaction of CheY proteins with CheA proteins and allowed rewiring of the signalling pathway. Mechanisms of signal integration and signal termination have been elucidated by mathematical modelling. Some bacteria have complex chemotaxis pathways that go beyond what is found in E. coli and R. sphaeroides . For example, in addition to the methylation-based adaptation system, Bacillus subtilis has two further adaptation pathways, one involving CheC and CheD and another using CheV. Some bacteria exploit the ability of the chemotaxis circuitry to sense small changes in ligand concentrations, and use the system to control behaviour other than chemotaxis. For example, Myxococcus xanthus has a chemotaxis-like pathway controlling development of the fruiting body, and Pseudomonas aeruginosa has one controlling biofilm formation. In this Review, Armitage and colleagues describe how some bacterial species, as typified by Rhodobacter sphaeroides , have evolved to contain complex chemotaxis signalling networks that integrate sensory information from the environment with metabolic information from within the cell to produce a balanced response at the flagellar motor. Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control swimming direction. This basic pathway is conserved across all chemotactic bacteria and archaea; however, recent work combining systems biology and genome sequencing has started to elucidate the additional complexity of the process in many bacterial species. This article focuses on one of the best understood complex networks, which is found in Rhodobacter sphaeroides and integrates sensory data about the external environment and the metabolic state of the cell to produce a balanced response at the flagellar motor.

Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal \"needle complex\" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

Most motile bacteria navigate within gradients of external chemical stimuli by regulating the length of randomly oriented swimming episodes. Magnetotactic bacteria are characterized by chains of intracellular ferromagnetic nanoparticles and their ability to sense the geomagnetic field, which is believed to facilitate directed motion, but is not well understood at the behavioural and molecular level. Here, we show that cells of Magnetospirillum gryphiswaldense unexpectedly display swimming polarity that depends on aerotactic signal transduction through one of its four chemotaxis operons ( cheOp1 ). Growth of cells in magnetic fields superimposed on oxygen gradients results in a gradual inherited bias of swimming runs with one of the cell poles leading, such that the resulting overall swimming direction of entire populations can be reversed by changes in oxygen concentration. These findings clearly show that there is a direct molecular link between aerotactic sensing and the determination of magnetotactic polarity, through the sensory pathway, CheOp1. Magnetotactic bacteria sense and migrate along the geomagnetic field, but the molecular mechanism for directed motion is not known. Here, Popp et al. show that M. gryphiswaldense displays swimming polarity in an oxygen gradient sensed by the chemotactic sensory pathway CheOp1, revealing a link between aerotactic sensing and magnetotactic polarity.

Spatial organization of signalling is not an exclusive property of eukaryotic cells. Despite the fact that bacterial signalling pathways are generally simpler than those in eukaryotes, there are several well‐documented examples of higher‐order intracellular signalling structures in bacteria. One of the most prominent and best‐characterized structures is formed by proteins that control bacterial chemotaxis. Signals in chemotaxis are processed by ordered arrays, or clusters, of receptors and associated proteins, which amplify and integrate chemotactic stimuli in a highly cooperative manner. Receptor clusters further serve to scaffold protein interactions, enhancing the efficiency and specificity of the pathway reactions and preventing the formation of signalling gradients through the cell body. Moreover, clustering can also ensure spatial separation of multiple chemotaxis systems in one bacterium. Assembly of receptor clusters appears to be a stochastic process, but bacteria evolved mechanisms to ensure optimal cluster distribution along the cell body for partitioning to daughter cells at division. Victor Sourjik and Judith Armitage outline the organization and differing complexity of chemotaxis pathways in Escherichia coli and Rhodobacter sphaeroides .

Many bacteria use a type III secretion system (T3SS) to inject effector proteins into host cells. Selection and export of the effectors is controlled by a set of soluble proteins at the cytosolic interface of the membrane spanning type III secretion ‘injectisome’. Combining fluorescence microscopy, biochemical interaction studies and fluorescence correlation spectroscopy, we show that in live Yersinia enterocolitica bacteria these soluble proteins form complexes both at the injectisome and in the cytosol. Binding to the injectisome stabilizes these cytosolic complexes, whereas the free cytosolic complexes, which include the type III secretion ATPase, constitute a highly dynamic and adaptive network. The extracellular calcium concentration, which triggers activation of the T3SS, directly influences the cytosolic complexes, possibly through the essential component SctK/YscK, revealing a potential mechanism involved in the regulation of type III secretion. Bacterial type III secretion systems (T3SS) play important roles in pathogenesis. Here, Diepold et al . show the dynamic nature of complexes formed of essential T3SS components in live bacteria, and that extracellular calcium concentrations influence these cytosolic complexes likely via SctK/YscK.

It is becoming clear that the bacterial flagellar motor output is important not only for bacterial locomotion but also for mediating the transition from liquid to surface living. The output of the flagellar motor changes with the mechanical load placed on it by the external environment: at a higher load, the motor runs more slowly and produces higher torque. Here we show that the number of torque-generating units bound to the flagellar motor also depends on the external mechanical load, with fewer stators at lower loads. Stalled motors contained at least as many stators as rotating motors at high load, indicating that rotation is unnecessary for stator binding. Mutant stators incapable of generating torque could not be detected around the motor. We speculate that a component of the bacterial flagellar motor senses external load and mediates the strength of stator binding to the rest of the motor. IMPORTANCE The transition between liquid living and surface living is important in the life cycles of many bacteria. In this paper, we describe how the flagellar motor, used by bacteria for locomotion through liquid media and across solid surfaces, is capable of adjusting the number of bound stator units to better suit the external load conditions. By stalling motors using external magnetic fields, we also show that rotation is not required for maintenance of stators around the motor; instead, torque production is the essential factor for motor stability. These new results, in addition to previous data, lead us to hypothesize that the motor stators function as mechanosensors as well as functioning as torque-generating units. The transition between liquid living and surface living is important in the life cycles of many bacteria. In this paper, we describe how the flagellar motor, used by bacteria for locomotion through liquid media and across solid surfaces, is capable of adjusting the number of bound stator units to better suit the external load conditions. By stalling motors using external magnetic fields, we also show that rotation is not required for maintenance of stators around the motor; instead, torque production is the essential factor for motor stability. These new results, in addition to previous data, lead us to hypothesize that the motor stators function as mechanosensors as well as functioning as torque-generating units.

The bacterial cytoplasm is known to be crowded, with that crowding suggested to change with growth, chromosome replication, and under stress conditions. Many physiological activities depend on proteins and substrates diffusing through the cytoplasm; in some cases, large complexes need to diffuse from pole to pole. The bacterial cytoplasm is a very crowded environment, and changes in crowding are thought to have an impact on cellular processes including protein folding, molecular diffusion and complex formation. Previous studies on the effects of crowding have generally compared cellular activity after imposition of stress. In response to different light intensities, in unstressed conditions, Rhodobacter sphaeroides changes the number of 50-nm intracytoplasmic membrane (ICM) vesicles, with the number varying from a few to over a thousand per cell. In this work, the effects of crowding induced by ICM vesicles in photoheterotrophic R. sphaeroides were investigated using a fluorescence resonance energy transfer (FRET) sensor and photoactivated localization microscopy (PALM). In low light grown cells where the cytoplasm has large numbers of ICM vesicles, the FRET probe adopts a more condensed conformation, resulting in higher FRET ratio readouts compared to high light cells with fewer ICM vesicles. The apparent diffusion coefficients of different sized proteins, PAmCherry, PAmCherry-CheY 6 , and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27 kDa decreased as the number of ICM vesicles increased. In low light R. sphaeroides where the crowding level is high, protein molecules were found to diffuse more slowly than in aerobic and high light cells. This suggests that some physiological activities might show different kinetics in bacterial species whose intracellular membrane organization can change with growth conditions. IMPORTANCE The bacterial cytoplasm is known to be crowded, with that crowding suggested to change with growth, with chromosome replication, and under stress conditions. Many physiological activities depend on proteins and substrates diffusing through the cytoplasm; in some cases, large complexes need to diffuse from pole to pole. It is unclear how increases in crowding might affect cellular functions. We investigated whether we could naturally change the crowded state of the Rhodobacter sphaeroides cytoplasm by growing under different growth conditions. We show that increasing the number of intracytoplasmic vesicles by growing photosynthetically does change the crowded state of the cytoplasm and also alters the diffusion rates of different sized proteins measured. As many other cellular processes require protein movement, these findings could have broader implications for bacterial growth and responses under changing conditions that could alter cytoplasmic crowding.

The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

The dynamic exchange of motor proteins in a functioning 45-nm rotary complex was observed in vivo and GFP–MotB stator proteins visualized in individual bacterial flagellar motors. Fluorescence recovery after photobleaching reveals that the ∼22 MotB motor proteins exchange rapidly with a pool of free protein while the motor is actively rotating. Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media 1 , 2 , 3 . Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall 4 , 5 . Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli . We monitored motor function by rotation of a tethered cell body 6 , and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP–MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP–MotB molecules diffusing at ∼0.008 µm 2  s -1 . Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s -1 : the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli . Our results confirm that, in vivo , FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. IMPORTANCE The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor. The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.