Feedback control architecture & the bacterial chemotaxis network

Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to ‘reset’ (adapt) the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a ‘cascade control’ feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance

Feedback control architecture and the bacterial chemotaxis network.

PMCID: PMC3088647This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to 'reset' (adapt) the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a 'cascade control' feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance

A Minimal Model of Metabolism Based Chemotaxis

Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis

The ParC/ParP system in the localization and segregation of chemotaxis signaling arrays in Vibrio cholerae and Vibrio parahaemolyticus

Chemotaxis proteins organize into large, highly ordered arrays. Particularly, in the enteric bacteria Vibrio cholerae and Vibrio parahaemolyticus, chemotaxis arrays are found at the cell pole, and their distribution follows a cell cycle dependent localization. The ParC/ParP system mediates this localization pattern and without either ParC or ParP, arrays are no longer positioned at the cell poles and fail to segregate upon division. Localization of arrays in these bacteria follow a hierarchical process, where arrays are tethered by ParP, which in turn links them to ParC, an ATPase that serves as a cell pole determinant in Vibrios. Here, we analyze the mechanism behind ParP’s ability to access the chemotaxis arrays and positions them at the cell pole. Furthermore, we show that even in the absence of histidine kinase CheA proteins, the arrays still exhibit the native spatial localization and the iconic hexagonal packing of the receptors. We show that the V. cholerae Cluster II array is versatile in respect of array composition for auxiliary chemotaxis proteins, such as ParP and that these arrays are structurally less stable due to their lower CheA occupancy in comparison to the ultrastable arrays found in E.coli. Additionally, we examine the dynamic localization of ParC and evaluate its influence in the overall localization of the arrays and ParP. We show that ParP’s C-terminus integrates into the core unit of signaling arrays through interactions with MCP proteins and the histidine kinase CheA. Our results indicate that ParP’s intercalation within the core units facilitates array formation, whereas its N-terminal interaction domain enables polar recruitment of arrays and promotes ParP’s own polar localization. Moreover, the data provides evidence that ParP serves as a critical nexus between the formation of the chemotactic arrays and their proper polar recruitment. Additionally, our data revealed that arrays in V. cholerae have the capacity to include several scaffolding proteins, displaying a previously uncharacterized variability. In turn, we demonstrate that this variability explains the high degree of structural instability shown by V. cholerae chemotaxis arrays. Finally, we show that ParC forms a protein gradient in V. parahaemolyticus cells. This protein gradient extends in a decreasing concentration from the cell pole towards mid-cell, and it is essential for ParC’s function in positioning ParP and consequently the chemosensory arrays. Similarly, gradient maintenance requires a continuous cycle of ParC between the cell pole and the cytoplasm, as well as ParC’s ability to associate with DNA and transition into different protein states in a nucleotide dependent manner. The data shows that ParC’s localization dynamics relies upon differential diffusion rates of its distinct protein states. Altogether, this work studies the complexity of the ParC/ ParP system and highlights the importance of each component in the correct placement of the chemotactic signaling arrays

Deciphering chemotaxis pathways using cross species comparisons

<p>Abstract</p> <p>Background</p> <p>Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. <it>Escherichia coli </it>utilises a single sensory pathway, but little is known about signalling pathways in species with more complex systems.</p> <p>Results</p> <p>To investigate whether chemotaxis pathways in other bacteria follow the <it>E. coli </it>paradigm, we analysed 206 species encoding at least 1 homologue of each of the 5 core chemotaxis proteins (CheA, CheB, CheR, CheW and CheY). 61 species encode more than one of all of these 5 proteins, suggesting they have multiple chemotaxis pathways. Operon information is not available for most bacteria, so we developed a novel statistical approach to cluster <it>che </it>genes into putative operons. Using operon-based models, we reconstructed putative chemotaxis pathways for all 206 species. We show that <it>cheA-cheW </it>and <it>cheR-cheB </it>have strong preferences to occur in the same operon as two-gene blocks, which may reflect a functional requirement for co-transcription. However, other <it>che </it>genes, most notably <it>cheY</it>, are more dispersed on the genome. Comparison of our operons with shuffled equivalents demonstrates that specific patterns of genomic location may be a determining factor for the observed <it>in vivo </it>chemotaxis pathways.</p> <p>We then examined the chemotaxis pathways of <it>Rhodobacter sphaeroides</it>. Here, the PpfA protein is known to be critical for correct partitioning of proteins in the cytoplasmically-localised pathway. We found <it>ppfA </it>in <it>che </it>operons of many species, suggesting that partitioning of cytoplasmic Che protein clusters is common. We also examined the apparently non-typical chemotaxis components, CheA3, CheA4 and CheY6. We found that though variants of CheA proteins are rare, the CheY6 variant may be a common type of CheY, with a significantly disordered C-terminal region which may be functionally significant.</p> <p>Conclusions</p> <p>We find that many bacterial species potentially have multiple chemotaxis pathways, with grouping of <it>che </it>genes into operons likely to be a major factor in keeping signalling pathways distinct. Gene order is highly conserved with <it>cheA-cheW </it>and <it>cheR-cheB </it>blocks, perhaps reflecting functional linkage. CheY behaves differently to other Che proteins, both in its genomic location and its putative protein interactions, which should be considered when modelling chemotaxis pathways.</p

Application of Computational Molecular Biophysics to Problems in Bacterial Chemotaxis

The combination of physics, biology, chemistry, and computer science constitutes the promising field of computational molecular biophysics. This field studies the molecular properties of DNA, protein lipids and biomolecules using computational methods. For this dissertation, I approached four problems involving the chemotaxis pathway, the set of proteins that function as the navigation system of bacteria and lower eukaryotes. In the first chapter, I used a special-purpose machine for molecular dynamics simulations, Anton, to simulate the signaling domain of the chemoreceptor in different signaling states for a total of 6 microseconds. Among other findings, this study provides enough evidence to propose a novel molecular mechanism for the kinase activation by the chemoreceptor and reconcile previously conflicting experimental data. In the second chapter, my molecular dynamics studies of the scaffold protein cheW reveals the existence and role of a conserved salt-bridge that stabilizes the relative position of the two binding sites in the chew surface: the chemoreceptor and the kinase. The results were further confirmed with NMR experiments performed with collaborators at the University of California in Santa Barbara, CA. In the third chapter, my colleagues and I investigate the quality of homology modeled structures with cheW protein as a benchmark. By subjecting the models to molecular dynamics and Monte Carlo simulations, we show that the homology models are snapshots of a larger ensemble of conformations very similar to the one generated by the experimental structures. In the fourth chapter, I use bioinformatics and basic mathematical modeling to predict the specific chemoreceptor(s) expressed in vivo and imaged with electron cryo tomography (ECT) by our collaborators at the California Institute of Technology. The study was essential to validate the argument that the hexagonal arrangement of transmembrane chemoreceptors is universal among bacteria, a major breakthrough in the field of chemotaxis. In summary, this thesis presents a collection of four works in the field of bacterial chemotaxis where either methods of physics or the quantitative approach of physicists were of fundamental importance for the success of the project

Analysis of cyclic di-GMP signaling components in "caulobacter crescentus" behavior and cell cycle control

Cell cycle progression and polar morphogenesis in Caulobacter crescentus are coordinated by the interplay of multiple proteins in time and space. One major regulatory factor is the second messenger cyclic di-GMP (c-di-GMP) therefore especially the activities of enzymes that are responsible for synthesis and breakdown of this small molecule are tightly regulated. The swarmer cell specific population in the early phase of the cell cycle contains low levels of c-di-GMP due to the action of the phosphodiesterase PdeA. During the course of cell cycle progression, PdeA is degraded and thereby the activity of the diguanylate cyclase (DGC) DgcB is released. At the same time a second DGC, PleD, is activated by a phosphorylation relay, to elevate c-di-GMP levels necessary for cell development. The two proteins DgcB and PleD are the main cyclases in C. crescentus contributing to the intracellular c-di-GMP pool. Cells lacking both DGCs have severe defects affecting cell morphology and cell cycle progression. However, a residual c-di-GMP concentration is still detectable in the pleD dgcB double mutant presumingly due to the activity of other DGCs of C. crescentus. This work addressed the question, which additional GGDEF domain proteins reveal DGC activity and contribute to the c-di-GMP content in C. crescentus cells. This work presented here shows that two additional cyclases, BipB and BipC (bifunctional proteins B and C), are involved in c-di-GMP signaling. Both enzymes belong to the group of so-called composite proteins harboring a GGDEF and EAL domain, encoding for opposing catalytic activities, respectively. Single deletions of either bipB or bipC showed no phenotype. However, in combination with the deletion of pleD and dgcB, no c-di-GMP could be detected. The lack of c-di-GMP resulted in miss-localization of the effector protein PopA that is involved in the degradation of the replication inhibitor CtrA. Therefore, CtrA is stabilized in those cells leading to elongated cell morphology. These phenotypes resemble the phenotypes of a strain lacking all predicted DGCs (gutted strain, GS). To measure specifically low levels of c-di-GMP a strain was used lacking DGCs and in addition all PDEs (really gutted strain, rGS) to avoid immediate degradation in the GS. Introduction of either bipB or bipC in the rGS reverted the strain to a wild-type phenotype, e.g. motility and popA localization, indicating a DGC phenotype in vivo. However, in the presence of different PDEs like in the GS neither bipB nor bipC were able to revert the phenotype to wild-type suggesting weak DGC activity of both enzymes. For BipB bifunctional enzyme activity could be demonstrated in vitro and in vivo, whereas the DGC and the PDE activities were present at the same time. The cyclase activity of BipB is substrate inhibited via c-di-GMP binding to the inhibitory site motif RxxD. Based on these finding we propose that BipB is a bifunctional protein contributing under the applied conditions with BipC, PleD and DgcB to intracellular c-di-GMP levels in C. crescentus. The c-di-GMP signaling circuit involves not only cyclases and phosphodiesterases, which produce c-di-GMP upon an environmental stimulus but also effector proteins that bind c-di-GMP and therefore transmit the signal into an intracellular response. Knowing different c-di-GMP binding proteins would allow understanding c-di-GMP output systems. Therefore, a biochemical screen was carried out using c-di-GMP linked to a capture compound to specifically isolate c-di-GMP binding proteins. Among the novel identified proteins a group clusters next to chemotaxis genes. One of the hits is CmcA (named after its involvement in c-di-GMP dependent motor control), a single domain response regulator lacking the conserved phosphorylation site (aspartate) necessary for the function of a RR. Deletion of cmcA results in an increase in motility. To transmit the chemotactic signal CheY proteins interact directly with the flagellar apparatus. Therefore, the localization pattern of CmcA in different flagellar mutants was determined showing polar localization dependent on the MS-ring forming protein FliF. This localization pattern is missing in c-di-GMP deficient cells. From these results, we concluded that CmcA regulates motility in a c�di-GMP dependent manner

Investigating the Function and the Interaction Network of the Flagellar Regulator ATPase FlhG

Motility plays a key role for the superior survival strategy of many bacteria. Sophisticated, macromolecular machines, called flagella, serve as bacterial locomotion organelles. These flagella appear in distinct spatial arrangements along the bacterial cell, constituting the flagellation patterns, whose disruption is detrimental to motility. However, the number of flagellation patterns that have arisen in a plethora of bacterial species can be counted by the fingers of one hand. How these patterns are established in the first place, and how they are maintained during cell division, remains a yet unassessed task in the field. Two nucleotide-binding proteins, FlhF and FlhG, were identified to be crucial for the spatial regulation of flagella in most flagellated bacteria, which exhibit various flagellation patterns. This work presents a structural and biochemical characterization of the flagella regulating ATPase FlhG, which revealed its function as a molecular switch, having a dimeric, membrane-associated state and a mobile, monomeric state in the cytoplasm. This hallmark feature of MinD/ParA ATPases is conserved in FlhG of peritrichous B. subtilis as well as monotrichous S. putrefaciens. In both organisms FlhG interacts with the flagellar C-ring components FliM and FliN(Y) providing insight into its role as a flagellar C-ring assembly factor, coordinating the assembly of a FliM/FliN(Y) complex to FliG. Differences in the regulatory networks underlying different flagellation patterns were identified in species-specific interaction partners of FlhG, such as the flagellar master regulator FlrA in S. putrefaciens or the late divisome component GpsB in B. subtilis. These findings led to the hypothesis that the spatial arrangement of flagella is encoded in the structure of the interaction network of FlhF and FlhG. This hypothesis is supported by the occurrence of varying C-ring components in differently flagellated bacteria. This work also includes the implementation and successful application of 1H/2H exchange mass spectrometry in Marburg. Not only does this powerful tool allow the convenient investigation of protein dynamics, but also the rapid mapping of protein-protein and protein-ligand interfaces. Interface mapping, in particular, revealed the power of this method and was applied in various research projects