Publications by year
In Press
Fadel F, Bassim V, Francis V, Porter S, Botzanowski T, Legrand P, Perez M, Bourne Y, Cianferani S, Vincent F, et al (In Press). Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS. Structure
Francis VI, Porter SL (In Press). Multikinase Networks: Two-Component Signaling Networks Integrating Multiple Stimuli. Annual Review of Microbiology, 73
2021
Brinkman FSL, Winsor GL, Done RE, Filloux A, Francis VI, Goldberg JB, Greenberg EP, Han K, Hancock REW, Haney CH, et al (2021). The Pseudomonas aeruginosa whole genome sequence: a 20th anniversary celebration. In (Ed)
Advances in Microbial Physiology, 25-88.
Abstract:
The Pseudomonas aeruginosa whole genome sequence: a 20th anniversary celebration
Abstract.
2020
Plume J (2020). Characterising a novel heavy metal-sensing multikinase network in the environmental organism and opportunistic pathogen, Burkholderia cenocepacia.
Abstract:
Characterising a novel heavy metal-sensing multikinase network in the environmental organism and opportunistic pathogen, Burkholderia cenocepacia
Two-component systems (TCSs) are a primary means of responding to environmental cues across the bacterial domain, and usually act independently of one another, forming discrete, isolated units. Multikinase networks (MKNs) are systems of multiple TCSs which can interact to integrate numerous signals into a decided cellular output, conferring an advantage to the cell. This work focuses on a novel MKN from the opportunistic pathogen, Burkholderia cenocepacia.
Through purification of each putative TCS, their ability to undergo non-cognate phosphorylation with one another was assessed in vitro through the phosphotransfer assay. This revealed that three sensor kinases, BCAM0442, BCAM0715 and BCAS0585 are able to phosphorylate one another’s response regulators. This non-cognate phosphotransfer was also possible, albeit to a lesser extent, in the presence of the cognate response regulator protein.
Deletion mutants of each TCS and investigation of TCS promoter induction revealed that BCAM0442/3 is implicated in copper resistance, and BCAM0714/5 is implicated in cadmium and zinc resistance. Exposure of B. cenocepacia to copper or zinc enhances resistance to imipenem, a phenomenon in which this MKN is implicated. A MKN deletion mutant is heavily attenuated in virulence in Galleria mellonella, linking the metal response with virulence in B. cenocepacia. Additionally, exploration of the copper response by RNA-seq analysis revealed substantial upregulation of the genes surrounding BCAM0442/3. A CopABCDE-like system was strongly upregulated in a BCAM0442/3-dependent manner, suggesting that BCAM0442/3 directly regulates this system.
Previous work identified that BCAM0714/5 regulates a downstream gene region, BCAM0716-21, deletion of which confers susceptibility to zinc in B. cenocepacia. Complementation with the BCAM0716/17 gene region restored resistance to zinc, implicating these genes in the zinc response of B. cenocepacia.
Given the environmental impact of heavy metal pollution and the use of metals as both an antibacterial strategy by the immune system and in medical devices, understanding the mechanisms of bacterial metal resistance is vital. This work has identified a novel metal-sensing MKN in B. cenocepacia, linking the cadmium, zinc and copper responses with virulence and carbapenem resistance.
Abstract.
Hubert C (2020). Characterising the role of Vibrio vulnificus type 6 secretion systems 1 and 2 in an in vivo oyster model.
Abstract:
Characterising the role of Vibrio vulnificus type 6 secretion systems 1 and 2 in an in vivo oyster model
Vibrio vulnificus is a significant human pathogen commonly isolated from temperate marine environments, where it is particularly abundant within filter-feeding shellfish. V. vulnificus is currently increasing in prevalence, theorised to be due to climate change facilitating V. vulnificus growth in previously inhospitable environments. Infection of susceptible individuals with V. vulnificus typically results in either primary septicaemia or necrotic wound infection, depending upon the route of entry, and frequently results in death if not treated rapidly.
Two type 6 secretion systems (T6SS) have been identified in V. vulnificus, termed the T6SS1 and the T6SS2. The T6SS is a molecular syringe utilised to inject cytotoxic effector proteins into neighbouring cells. Whilst the T6SS2 is present in all sequenced
V. vulnificus strains, only a subset possesses the T6SS1. Previous bacterial co-culture killing assays between T6SS1+ and T6SS1- V. vulnificus strains demonstrated thermoregulated T6SS1-mediated killing of T6SS1- strains. This study further characterised the role of both the T6SS1 and the T6SS2 in vitro. In vitro co-culture assays demonstrated that both the T6SS1 and the T6SS2 have antibacterial killing activity at the environmentally representative temperature of 21 °C. This is the first characterised role for the T6SS2 of V. vulnificus. No anti-eukaryotic activity was observed following co-culture with the phagocytic amoeba, Dictyostelium discoideum, suggesting that T6SS activity is purely antibacterial.
In vitro bacterial co-culture assays were replicated in vivo using an oyster model. To facilitate high-level uptake of bacterial strains of interest by oysters, an artificial marine snow model was developed where bacteria were incorporated into easily ingested phytoplankton aggregates. Uptake of bacteria from artificial marine snow was extremely successful, resulting in bacterial loads within oysters significantly greater than achieved by any study to date. Using this model, this study was able to demonstrate that V. vulnificus utilises both the T6SS1 and the T6SS2 to target and kill neighbouring bacteria, in both an intra and inter-species manner. This data suggests that the T6SSs of V. vulnificus play a key role in V. vulnificus ecology and the dynamics between bacterial populations in vivo.
Abstract.
2019
Lowry RC, Milner DS, Al-Bayati AMS, Lambert C, Francis VI, Porter SL, Sockett RE (2019). Evolutionary diversification of the RomR protein of the invasive deltaproteobacterium, Bdellovibrio bacteriovorus.
Sci Rep,
9(1).
Abstract:
Evolutionary diversification of the RomR protein of the invasive deltaproteobacterium, Bdellovibrio bacteriovorus.
Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that encounters individual Gram-negative prey bacteria with gliding or swimming motility, and then is able to invade such prey cells via type IVa pilus-dependent mechanisms. Movement control (pili or gliding) in other deltaproteobacteria, such as the pack hunting Myxococcus xanthus, uses a response regulator protein, RomRMx (which dynamically relocalises between the cell poles) and a GTPase, MglAMx, previously postulated as an interface between the FrzMx chemosensory system and gliding or pilus-motility apparatus, to produce regulated bidirectional motility. In contrast, B. bacteriovorus predation is a more singular encounter between a lone predator and prey; contact is always via the piliated, non-flagellar pole of the predator, involving MglABd, but no Frz system. In this new study, tracking fluorescent RomRBd microscopically during predatory growth shows that it does not dynamically relocalise, in contrast to the M. xanthus protein; instead having possible roles in growth events. Furthermore, transcriptional start analysis, site-directed mutagenesis and bacterial two-hybrid interaction studies, indicate an evolutionary loss of RomRBd activation (via receiver domain phosphorylation) in this lone hunting bacterium, demonstrating divergence from its bipolar role in motility in pack-hunting M. xanthus and further evolution that may differentiate lone from pack predators.
Abstract.
Author URL.
2018
Francis V, Waters EM, Finton-James SE, Gori A, Kadioglu A, Brown A, Porter SL (2018). Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa. Nature Communications, 9, 2219-2219.
2017
Francis VI, Stevenson EC, Porter SL (2017). Two-component systems required for virulence in Pseudomonas aeruginosa.
FEMS Microbiology Letters,
364(11).
Abstract:
Two-component systems required for virulence in Pseudomonas aeruginosa
Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP.
Abstract.
2014
Amin M, Kothamachu VB, Feliu E, Scharf BE, Porter SL, Soyer OS (2014). Phosphate sink containing two-component signaling systems as tunable threshold devices.
PLoS Comput Biol,
10(10).
Abstract:
Phosphate sink containing two-component signaling systems as tunable threshold devices.
Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications.
Abstract.
Author URL.
2013
Amin M, Porter SL, Soyer OS (2013). Split histidine kinases enable ultrasensitivity and bistability in two-component signaling networks.
PLoS Comput Biol,
9(3).
Abstract:
Split histidine kinases enable ultrasensitivity and bistability in two-component signaling networks.
Bacteria sense and respond to their environment through signaling cascades generally referred to as two-component signaling networks. These networks comprise histidine kinases and their cognate response regulators. Histidine kinases have a number of biochemical activities: ATP binding, autophosphorylation, the ability to act as a phosphodonor for their response regulators, and in many cases the ability to catalyze the hydrolytic dephosphorylation of their response regulator. Here, we explore the functional role of "split kinases" where the ATP binding and phosphotransfer activities of a conventional histidine kinase are split onto two distinct proteins that form a complex. We find that this unusual configuration can enable ultrasensitivity and bistability in the signal-response relationship of the resulting system. These dynamics are displayed under a wide parameter range but only when specific biochemical requirements are met. We experimentally show that one of these requirements, namely segregation of the phosphatase activity predominantly onto the free form of one of the proteins making up the split kinase, is met in Rhodobacter sphaeroides. These findings indicate split kinases as a bacterial alternative for enabling ultrasensitivity and bistability in signaling networks. Genomic analyses reveal that up 1.7% of all identified histidine kinases have the potential to be split and bifunctional.
Abstract.
Author URL.
2011
Porter SL, Wilkinson DA, Byles ED, Wadhams GH, Taylor S, Saunders NJ, Armitage JP (2011). Genome sequence of Rhodobacter sphaeroides Strain WS8N.
J Bacteriol,
193(15), 4027-4028.
Abstract:
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.
Abstract.
Author URL.
Porter SL, Wadhams GH, Armitage JP (2011). Signal processing in complex chemotaxis pathways.
Nat Rev Microbiol,
9(3), 153-165.
Abstract:
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.
Abstract.
Author URL.
2010
Tindall MJ, Porter SL, Maini PK, Armitage JP (2010). Modeling chemotaxis reveals the role of reversed phosphotransfer and a bi-functional kinase-phosphatase.
PLoS Comput Biol,
6(8).
Abstract:
Modeling chemotaxis reveals the role of reversed phosphotransfer and a bi-functional kinase-phosphatase.
Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY(6), aided by its specific phosphatase, the bifunctional kinase CheA(3), acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB(2) connects the cytoplasmic cluster kinase CheA(3) with the polar localised kinase CheA(2), and allows CheA(3)-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA(3) to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.
Abstract.
Author URL.
Scott KA, Porter SL, Bagg EAL, Hamer R, Hill JL, Wilkinson DA, Armitage JP (2010). Specificity of localization and phosphotransfer in the CheA proteins of Rhodobacter sphaeroides.
Molecular MicrobiologyAbstract:
Specificity of localization and phosphotransfer in the CheA proteins of Rhodobacter sphaeroides
Specificity of protein–protein interactions plays a vital role in signal transduction. The chemosensory pathway of Rhodobacter sphaeroides comprises multiple homologues of chemotaxis proteins characterized in organisms such as Escherichia coli. Three CheA homologues are essential for chemotaxis in R. sphaeroides under laboratory conditions. These CheAs are differentially localized to two chemosensory
clusters, one at the cell pole and one in the
cytoplasm. The polar CheA, CheA2, has the same
domain structure as E. coli CheA and can phosphorylate all R. sphaeroides chemotaxis response regulators. CheA3 and CheA4 independently localize to the cytoplasmic cluster; each protein has a subset of the CheA domains, with CheA3 phosphorylating CheA4 together making a functional CheA protein.
Interestingly, CheA3-P can only phosphorylate two
response regulators, CheY6 and CheB2. R. sphaeroides CheAs exhibit two interesting differences in specificity: (i) the response regulators that they phosphorylate and (ii) the chemosensory cluster to which they localize. Using a domain-swapping approach we investigated the role of the P1 and P5 CheA domains in determining these specificities. We show that the
P1 domain is sufficient to determine which response regulators will be phosphorylated in vitro while the P5 domain is sufficient to localize the CheAs to a specific chemosensory cluster.
Abstract.
Bell CH, Porter SL, Strawson A, Stuart DI, Armitage JP (2010). Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex.
PLoS Biol,
8(2).
Abstract:
Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex.
Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 a crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.
Abstract.
Author URL.
2009
Ind AC, Porter SL, Brown MT, Byles ED, de Beyer JA, Godfrey SA, Armitage JP (2009). Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans.
Appl Environ Microbiol,
75(20), 6613-6615.
Abstract:
Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans.
We have developed a stable isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI(q) gene from pYanni3 and the lac promoter, P(A1/04/03), from pJBA24.
Abstract.
Author URL.
Tindall MJ, Porter SL, Wadhams GH, Maini PK, Armitage JP (2009). Spatiotemporal modelling of CheY complexes in Escherichia coli chemotaxis.
Prog Biophys Mol Biol,
100(1-3), 40-46.
Abstract:
Spatiotemporal modelling of CheY complexes in Escherichia coli chemotaxis.
The chemotaxis pathway of Escherichia coli is one of the best studied and modelled biological signalling pathways. Here we extend existing modelling approaches by explicitly including a description of the formation and subcellular localization of intermediary complexes in the phosphotransfer pathway. The inclusion of these complexes shows that only about 60% of the total output response regulator (CheY) is uncomplexed at any moment and hence free to interact with its target, the flagellar motor. A clear strength of this model is its ability to predict the experimentally observable subcellular localization of CheY throughout a chemotactic response. We have found good agreement between the model output and experimentally determined CheY localization patterns.
Abstract.
Author URL.
2008
Porter SL, Roberts MAJ, Manning CS, Armitage JP (2008). A bifunctional kinase-phosphatase in bacterial chemotaxis.
Proc Natl Acad Sci U S A,
105(47), 18531-18536.
Abstract:
A bifunctional kinase-phosphatase in bacterial chemotaxis.
Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.
Abstract.
Author URL.
Tindall MJ, Porter SL, Maini PK, Gaglia G, Armitage JP (2008). Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell.
Bull Math Biol,
70(6), 1525-1569.
Abstract:
Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell.
Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area.
Abstract.
Author URL.
Tindall MJ, Maini PK, Porter SL, Armitage JP (2008). Overview of mathematical approaches used to model bacterial chemotaxis II: bacterial populations.
Bull Math Biol,
70(6), 1570-1607.
Abstract:
Overview of mathematical approaches used to model bacterial chemotaxis II: bacterial populations.
We review the application of mathematical modeling to understanding the behavior of populations of chemotactic bacteria. The application of continuum mathematical models, in particular generalized Keller-Segel models, is discussed along with attempts to incorporate the microscale (individual) behavior on the macroscale, modeling the interaction between different species of bacteria, the interaction of bacteria with their environment, and methods used to obtain experimentally verified parameter values. We allude briefly to the role of modeling pattern formation in understanding collective behavior within bacterial populations. Various aspects of each model are discussed and areas for possible future research are postulated.
Abstract.
Author URL.
Porter SL, Wadhams GH, Armitage JP (2008). Rhodobacter sphaeroides: complexity in chemotactic signalling.
Trends Microbiol,
16(6), 251-260.
Abstract:
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.
Abstract.
Author URL.
2007
Porter SL, Wadhams GH, Armitage JP (2007). In vivo and in vitro analysis of the Rhodobacter sphaeroides chemotaxis signaling complexes.
Methods Enzymol,
423, 392-413.
Abstract:
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.
Abstract.
Author URL.
2006
Slovak PM, Porter SL, Armitage JP (2006). Differential localization of Mre proteins with PBP2 in Rhodobacter sphaeroides.
J Bacteriol,
188(5), 1691-1700.
Abstract:
Differential localization of Mre proteins with PBP2 in Rhodobacter sphaeroides.
In Rhodobacter sphaeroides, MreB, MreC, MreD, PBP2, and RodA are encoded at the same locus. The localizations of PBP2, MreB, and MreC, which have all been implicated in the synthesis of the peptidoglycan layer, were investigated under different growth conditions to gain insight into the relationships between these proteins. Immunofluorescence microscopy showed that PBP2 localized to specific sites at the midcell of elongating cells under both aerobic and photoheterotrophic conditions. Visualizing PBP2 at different stages of the cell cycle showed that in elongating cells, PBP2 was found predominately at the midcell, with asymmetric foci and bands across the cell. PBP2 remained at midcell until the start of septation, after which it moved to midcell of the daughter cells. Deconvolution and three-dimensional reconstructions suggested that PBP2 forms a partial ring at the midcell of newly divided cells and elongated cells, while in septating cells, partial PBP2 rings were present at one-quarter and three-quarter positions. Due to the diffraction limits of light microscopy, these partial rings could represent unresolved helices. Colocalization studies showed that MreC always colocalized with PBP2, while MreB colocalized with PBP2 only during elongation; during septation, MreB remained at the septation site, whereas PBP2 relocalized to the one-quarter and three-quarter positions. These results suggest that PBP2 and MreC are involved in peptidoglycan synthesis during elongation and that this occurs at specific sites close to midcell in R. sphaeroides.
Abstract.
Author URL.
Rasmussen AA, Wegener-Feldbrügge S, Porter SL, Armitage JP, Søgaard-Andersen L (2006). Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity.
Mol Microbiol,
60(2), 525-534.
Abstract:
Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity.
In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.
Abstract.
Author URL.
Porter SL, Wadhams GH, Martin AC, Byles ED, Lancaster DE, Armitage JP (2006). The CheYs of Rhodobacter sphaeroides.
J Biol Chem,
281(43), 32694-32704.
Abstract:
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.
Abstract.
Author URL.
2005
Rasmussen AA, Porter SL, Armitage JP, Søgaard-Andersen L (2005). Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus.
Mol Microbiol,
56(5), 1358-1372.
Abstract:
Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus.
We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.
Abstract.
Author URL.
2004
Porter SL, Armitage JP (2004). Chemotaxis in Rhodobacter sphaeroides requires an atypical histidine protein kinase.
J Biol Chem,
279(52), 54573-54580.
Abstract:
Chemotaxis in Rhodobacter sphaeroides requires an atypical histidine protein kinase.
Rhodobacter sphaeroides has a complex chemosensory system comprising two classic CheAs, two atypical CheAs, and eight response regulators (six CheYs and two CheBs). The classic CheAs, CheA(1) and CheA(2), have similar domain structures to Escherichia coli CheA, whereas the atypical CheAs, CheA(3) and CheA(4), lack some of the domains found in E. coli CheA. CheA(2), CheA(3), and CheA(4) are all essential for chemotaxis. Here we demonstrate that CheA(3) and CheA(4) are both unable to undergo ATP-dependent autophosphorylation, however, CheA(4) is able to phosphorylate CheA(3). The in vitro kinetics of this phosphorylation reaction were consistent with a reaction mechanism in which CheA(3) associates with a CheA(4) dimer forming a complex, CheA(3)A(4). To the best of our knowledge, CheA(3)A(4) is the first characterized histidine protein kinase where the subunits are encoded by distinct genes. Selective phosphotransfer was observed from CheA(3)-P to the response regulators CheY(1), CheY(6), and CheB(2). Using phosphorylation site and kinase domain mutants of CheA we show that phosphosignaling involving CheA(2), CheA(3), and CheA(4) is essential for chemotaxis in R. sphaeroides. Interestingly, CheA(3) was not phosphorylated in vitro by CheA(1) or CheA(2), although CheA(1) and CheA(2) mutants with defective kinase domains were phosphorylated by CheA(4). Because in vivo CheA(3) and CheA(4) localize to the cytoplasmic chemotaxis cluster, while CheA(2) localizes to the polar chemotaxis cluster, it is likely that the physical separation of CheA(2) and CheA(4) prevents unwanted cross-talk between these CheAs.
Abstract.
Author URL.
2002
Porter SL, Armitage JP (2002). Phosphotransfer in Rhodobacter sphaeroides chemotaxis.
J Mol Biol,
324(1), 35-45.
Abstract:
Phosphotransfer in Rhodobacter sphaeroides chemotaxis.
The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R.sphaeroides has these multiple homologues. CheA(1) and CheA(2) contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA(2), and both are capable of ATP-dependent autophosphorylation. While the K(m) values for ATP of CheA(1) and CheA(2) were similar to that of E.coli, the k(cat) value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA(2) phosphorylated all of the CheYs and both CheBs, whilst CheA(1) did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY(1), CheY(2), and CheY(5)) and CheY(3). The dephosphorylation rates of the R.sphaeroides CheBs were much slower than the E.coli CheB. The dephosphorylation rate of CheY(6), encoded by the third chemosensory locus, was ten times faster than that of the E.coli CheY. However, the dephosphorylation rates of the remaining R.sphaeroides CheYs were comparable to that of E.coli CheY.
Abstract.
Author URL.
Porter SL, Warren AV, Martin AC, Armitage JP (2002). The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis.
Mol Microbiol,
46(4), 1081-1094.
Abstract:
The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis.
The purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.
Abstract.
Author URL.
Wadhams GH, Martin AC, Porter SL, Maddock JR, Mantotta JC, King HM, Armitage JP (2002). TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm.
Mol Microbiol,
46(5), 1211-1221.
Abstract:
TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm.
TlpC is encoded in the second chemotaxis operon of Rhodobacter sphaeroides. This protein shows some homology to membrane-spanning chemoreceptors of many bacterial species but, unlike these, is essential for R. sphaeroides chemotaxis to all compounds tested. Genomic replacement of tlpC with a C-terminal gfp fusion demonstrated that TlpC localized to a discrete cluster within the cytoplasm. Immunogold electron microscopy also showed that TlpC localized to a cytoplasmic electron-dense region. Correct TlpC-GFP localization depended on the downstream signalling proteins, CheW3, CheW4 and CheA2, and was tightly linked to cell division. Newly divided cells contained a single cluster but, as the cell cycle progressed, a second cluster appeared close to the initial cluster. As elongation continued, these clusters moved apart so that, on septation, each daughter cell contained a single TlpC cluster. The data presented suggest that TlpC is either a cytoplasmic chemoreceptor responding to or integrating global signals of metabolic state or a novel and essential component of the chemotaxis signalling pathway. These data also suggest that clustering is essential for signalling and that a mechanism may exist for targeting and localizing proteins within the bacterial cytoplasm.
Abstract.
Author URL.
2001
Martin AC, Wadhams GH, Shah DS, Porter SL, Mantotta JC, Craig TJ, Verdult PH, Jones H, Armitage JP (2001). CheR- and CheB-dependent chemosensory adaptation system of Rhodobacter sphaeroides.
J Bacteriol,
183(24), 7135-7144.
Abstract:
CheR- and CheB-dependent chemosensory adaptation system of Rhodobacter sphaeroides.
Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These include cheA(1) and cheR(1) (che Op(1)) and cheA(2), cheR(2), and cheB(1) (che Op(2)). In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR(2) and CheB(1) were essential for normal chemotaxis, whereas CheR(1) was not. cheR(2) and cheB(1), but not cheR(1), were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR(2) either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA(2), but not CheA(1), is able to phosphorylate CheB(1), suggesting that signaling through CheA(1) cannot initiate feedback receptor adaptation via CheB(1)-P.
Abstract.
Author URL.
2000
Shah DS, Porter SL, Martin AC, Hamblin PA, Armitage JP (2000). Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes.
EMBO J,
19(17), 4601-4613.
Abstract:
Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes.
Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators. The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response. CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom.
Abstract.
Author URL.
Shah DS, Porter SL, Harris DC, Wadhams GH, Hamblin PA, Armitage JP (2000). Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway.
Mol Microbiol,
35(1), 101-112.
Abstract:
Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway.
The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.
Abstract.
Author URL.