Dichotomous feedback: a signal sequestration-based feedback mechanism for biocontroller design.

We introduce a new design framework for implementing negative feedback regulation in synthetic biology, which we term 'dichotomous feedback'. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the process's architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realize than 'molecular sequestration' and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where a second response regulator could be competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

Use of transcriptomic data for extending a model of the AppA/PpsR system in Rhodobacter sphaeroides.

BACKGROUND: Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions. RESULTS: Our extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability. CONCLUSION: We found that despite at least three pathways being involved in the regulation of photosynthetic genes, the AppA/PpsR system alone is capable of accounting for the observed trends in photosynthetic gene expression seen at different oxygen levels.

A single phosphatase can convert a robust step response into a graded, tunable or adaptive response.

Many biological signalling pathways have evolved to produce responses to environmental signals that are robust to fluctuations in protein copy number and noise. Whilst beneficial for biology, this robustness can be problematic for synthetic biologists wishing to re-engineer and subsequently tune the response of a given system. Here we show that the well-characterized EnvZ/OmpR two-component signalling system from Escherichia coli possesses one such robust step response. However, the synthetic addition of just a single component into the system, an extra independently controllable phosphatase, can change this behaviour to become graded and tunable, and even show adaptation. Our approach introduces a new design principle which can be implemented simply in engineering and redesigning fast signal transduction pathways for synthetic biology.

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides.

Chemotaxis is one of the best-characterized signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterized the properties of the chemosensory response of Rhodobacter sphaeroides, an α-proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays, we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber's Law. Mathematical modelling also shows that, as E. coli, R. sphaeroides is capable of showing fold-change detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber's Law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

Predicting inter-species cross-talk in two-component signalling systems.

Phosphosignalling pathways are an attractive option for the synthetic biologist looking for a wide repertoire of modular components from which to build. We demonstrate that two-component systems can be used in synthetic biology. However, their potential is limited by the fact that host cells contain many of their own phosphosignalling pathways and these may interact with, and cross-talk to, the introduced synthetic components. In this paper we also demonstrate a simple bioinformatic tool that can help predict whether interspecies cross-talk between introduced and native two-component signalling pathways will occur and show both in vitro and in vivo that the predicted interactions do take place. The ability to predict potential cross-talk prior to designing and constructing novel pathways or choosing a host organism is essential for the promise that phosphosignalling components hold for synthetic biology to be realised.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes.

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.

Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides.

The FlgM secretion checkpoint plays a crucial role in coordinating bacterial flagellar assembly. Here we identify a new role for FlgM and FliA as part of a complex regulatory network which controls flagellum number and is essential for efficient swimming and biofilm formation in the monotrichous bacterium Rhodobacter sphaeroides.

Genome sequence of Rhodobacter sphaeroides Strain WS8N.

Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.

New motion analysis system for characterization of the chemosensory response kinetics of Rhodobacter sphaeroides under different growth conditions.

We developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers of Rhodobacter sphaeroides cells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured. Aerobically and photoheterotrophically grown R. sphaeroides exhibited significantly different chemosensory response kinetics and cell-to-cell variability in their responses to 100 µM propionate. A greater proportion of the population of aerobically grown cells responded to a 100 µM step decrease in propionate; they adapted faster and showed less cell-to-cell variability than photosynthetic populations. Growth in chemostats did not significantly reduce the measured cell to cell variability but did change the adaptation kinetics for photoheterotrophically grown cells.

Signal processing in complex chemotaxis pathways.

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.

Signal-dependent turnover of the bacterial flagellar switch protein FliM.

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the approximately 30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Rhodobacter sphaeroides: complexity in chemotactic signalling.

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli.

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at approximately 2200 pN nm) from stall up to a "knee" speed of approximately 420 Hz, and then falls linearly with speed, extrapolating to zero torque at approximately 910 Hz. Motors containing one to five stators generated approximately 200 pN nm per stator at speeds up to approximately 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to approximately 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to approximately 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.

In vivo and in vitro analysis of the Rhodobacter sphaeroides chemotaxis signaling complexes.

This chapter describes both the in vivo and in vitro methods that have been successfully used to analyze the chemotaxis pathways of R. sphaeroides, showing that two operons each encode a complete chemosensory pathway with each forming into independent signaling clusters. The methods used range from in vitro analysis of the chemotaxis phosphorylation reactions to protein localization experiments. In vitro analysis using purified proteins shows a complex pattern of phosphotransfer. However, protein localization studies show that the R. sphaeroides chemotaxis proteins are organized into two distinct sensory clusters -- one containing transmembrane receptors located at the cell poles and the other containing soluble chemoreceptors located in the cytoplasm. Signal outputs from both clusters are essential for chemotaxis. Each cluster has a dedicated chemotaxis histidine protein kinase (HPK), CheA. There are a total of eight chemotaxis response regulators in R. sphaeroides, six CheYs and two CheBs, and each CheA shows a different pattern of phosphotransfer to these response regulators. The spatial separation of homologous proteins may mean that reactions that happen in vitro do not occur in vivo, suggesting great care should be taken when extrapolating from purely in vitro data to cell physiology. The methods described in this chapter are not confined to the study of R. sphaeroides chemotaxis but are applicable to the study of complex two-component systems in general.

The CheYs of Rhodobacter sphaeroides.

The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues. The roles and cellular localization of the CheYs in Rhodobacter sphaeroides were determined. Only two CheYs were required for chemotaxis, CheY(6) and either CheY(3) or CheY(4). These CheYs were partially localized to either of the two chemotaxis signaling clusters, with the remaining protein delocalized. Interestingly, mutation of the CheY(6) phosphorylatable aspartate to asparagine produced a stopped motor, caused by phosphorylation on alternative site Ser-83 by CheA. Extensive mutagenesis of E. coli CheY has identified a number of activating mutations, which have been extrapolated to other response regulators (D13K, Y106W, and I95V). Analogous mutations in R. sphaeroides CheYs did not cause activation. These results suggest that although the R. sphaeroides and E. coli CheYs are similar in that they require phosphorylation for activation, they may differ in both the nature of the phosphorylation-induced conformational change and their subsequent interactions with the flagellar motor. Caution should therefore be used when projecting from E. coli CheY onto novel response regulators.

Stoichiometry and turnover in single, functioning membrane protein complexes.

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. 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. 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, 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 approximately 22 copies of GFP-MotB, consistent with approximately 11 stators each containing two MotB molecules. We also observed a membrane pool of approximately 200 GFP-MotB molecules diffusing at approximately 0.008 microm2 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 approximately 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

The positioning of cytoplasmic protein clusters in bacteria.

Cell division is a carefully orchestrated procedure. Bacterial cells have intricate mechanisms to ensure that genetic material is copied, proofread, and accurately partitioned into daughter cells. Partitioning now appears to also occur for some cytoplasmic proteins. Previously, using chromosomal fluorescent protein fusions, we demonstrated that a subset of Rhodobacter sphaeroides chemotaxis proteins colocalize to a discrete region within the bacterial cytoplasm. Using TlpT-yellow fluorescent protein as a marker for the position of the cytoplasmic protein clusters, we show most cells contain either one cluster localized at mid-cell or two clusters at the one-fourth and three-fourths positions of cell length. The number and positioning of these protein clusters depend on a previously unrecognized bacterial protein positioning factor, PpfA, which has homology to bacterial type I DNA partitioning factors. These data suggest that there is a mechanism involved in partitioning some cytoplasmic proteins upon cell division that is analogous to a mechanism seen for plasmid and chromosomal DNA.

Requirements for chemotaxis protein localization in Rhodobacter sphaeroides.

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli, the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.

Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure.

MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes.