Publications

Scholarly Journals--Published

• Stuffle E, Suzuki T, Orillard E, and Watts KJ. 2023. Aer2 signaling in Vibrio vulnificus with three PAS-heme domains. Mol Microbiol. 119(1):59-73.   (01/2023) (link)
• Anaya S, Orillard E, Greer-Phillips SE, and Watts KJ. 2022. New roles for HAMP domains: The tri-HAMP region of Pseudomonas aeruginosa Aer2 controls receptor signaling and cellular localization. J. Bacteriol. 204(9):e0022522.  (08/2022) (link)
• Orillard E, and Watts KJ. 2022. Leptospira interrogans Aer2: An unusual membrane-bound PAS-heme oxygen sensor. J. Bacteriol. 204(4):e0056721 (03/2022) (link)
• Orillard E, Anaya S, Johnson M, and Watts KJ. 2021. Oxygen-induced conformational changes in the PAS-heme domain of the Pseudomonas aeruginosa Aer2 receptor. Biochemistry. 60:2610-2622. The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain. (08/2021) (link)
• Stuffle E, Johnson MS, and Watts KJ. 2021. PAS domains in bacterial signal transduction. Curr Opin Microbiol.61:8-15. PAS domains are widespread, versatile domains found in proteins from all kingdoms of life. The PAS fold is composed of an antiparallel β-sheet with several flanking α-helices, and contains a conserved cleft for cofactor or ligand binding. The last few years have seen a prodigious increase in identified PAS domains and resolved PAS structures, including structures with effector and other domains. New bacterial PAS ligands have been discovered, and structure-function studies have improved our understanding of PAS signaling mechanisms. The list of bacterial PAS functions has now expanded to include roles in signal sensing, modulation, transduction, dimerization, protein interaction, and cellular localization. (06/2021) (link)
• Orillard E, and Watts KJ. 2021. Deciphering the Che2 chemosensory pathway and the roles of individual Che2 proteins from Pseudomonas aeruginosa. Mol Microbiol. 115:222-237.  Pseudomonas aeruginosa is an opportunistic pathogen that senses and responds to its environment via four chemosensory systems. Oxygen activates the Che2 chemosensory system by binding to the PAS-heme domain of the Aer2 receptor. Ostensibly, the output of Che2 occurs via its response regulator CheY2, but controversy persists over CheY2's exact role. In this study, we show that CheY2 does not interact with the flagellar motor and that the Che2 system does not transfer phosphoryl groups to the chemotaxis (Che) system. We show that CheY2 instead provides feedback control of Aer2 adaptation. In the presence of O2 , Aer2 signaling increases the autophosphorylation of the histidine kinase CheA2, followed by CheY2-mediated dephosphorylation. CheY2 does not stably retain phosphate and may not signal the output of the Che2 system. Rather, CheY2 activity enhances the direct interaction of CheY2 with the adaptation protein CheD (a role often facilitated by CheC, which P. aeruginosa lacks). In the absence of O2 , Aer2 does not signal, and CheY2/CheD interactions attenuate. This frees CheD to augment CheR2-mediated methylation of Aer2, which enhances Aer2 signaling. CheD does not interact with CheR2, but most likely interacts with Aer2 via conserved CheD-binding motifs to make Aer2 a better methylation substrate. (02/2021) (link)
• Ortega DR, Subramanian P, Mann P, Kjaer A, Chen S, Watts KJ, Pirbadian S, Collins DA, Kooger R, Kalyuzhnaya MG, Ringgaard S, Briegel A, and Jensen GJ. 2020. Repurposing a macromolecular machine: Architecture and evolution of the F7 chemosensory system. Nat Commun. 11, 2041. How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost. (04/2020) (link)
• Watts KJ, Vaknin A, Fuqua C, and Kazmierczak, BI. New twists and turns in bacterial locomotion and signal transduction. J. Bacteriol. Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo These exciting experimental advances were just some of the many novel findings presented at the 15thBacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019. (07/2019) (link)
• Greer-Phillips SE, Sukomon N, Khiang Chua E, Johnson MS, Crane BR and Watts KJ. 2018. The Aer2 receptor from Vibrio choleraeis a dual PAS-heme oxygen sensor. Mol Microbiol. 109(2):209-224. The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44-45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here we report that the cluster III receptor, VcAer2, binds and responds to O2 . VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2), but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O2 -sensor [Kd(O2) , 19 μM], utilizing the same Iβ Trp (W276) as PaAer2 to stabilize O2 . The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS, but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O2 [Kd(O2), 12 μM], although O2 binding was stabilized by either a Trp or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O2-mediated signaling from PAS2, and resulting in activation of the cluster III chemosensory pathway. (07/2018) (link)
• Greenswag AR, Li X, Borbat PP, Samanta D, Watts KJ, Freed JH and Crane BR. 2015. Preformed soluble chemoreceptor trimers that mimic cellular assembly states and activate CheA autophosphorylation. Biochemistry. 54:3454-3468 Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFOmodules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation. (05/2015) (link)
• Airola MV, Huh D, Sukomon N, Widom J, Sircar R, Borbat PP, Freed JH, Watts KJ, and Crane BR. 2013. Architecture of the soluble receptor Aer2 indicates an in-line mechanism for PAS and HAMP domain signaling. Journal of Molecular Biology. 425(5):886-901.  Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains. (03/2013) (link)
• Airola MV, Sukomon N, Samanta D, Borbat PP, Freed JH, Watts KJ, and Crane BR. 2013. HAMP domain conformers that propagate opposite signals in bacterial chemoreceptors. PLOS Biology. 11(2):e1001479. HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses. Paper: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001479 Commentary: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001480 BLOG: http://blogs.plos.org/biologue/2013/02/28/molecular-movements-drive-bacterial-signaling/ (02/2013) (link)
• Watts KJ, Johnson MS, and Taylor BL. 2011. Different conformations of the kinase-on and kinase-off signaling states in the Aer HAMP domain. J Bacteriol. 193(16):4095-103.  HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMPdiscontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMPbundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signalingmay be associated with a tilting of the AS-1 and AS-2' helices, which may be the signal that is transmitted to the kinase control region of Aer. (08/2011) (link)
• Watts KJ, Taylor BL, and Johnson MS. 2011. PAS/poly-HAMP signaling in Aer-2, a soluble heme-based sensor. Mol Microbiol. 79(3):686-699.  Poly-HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer-2, has an unusual domain architecture consisting of a PAS-sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains, followed by a conserved kinase control module. The structure of the N-terminal HAMP domains was recently solved, making Aer-2 the first protein with resolved poly-HAMP structure. The role of Aer-2 in P. aeruginosa is unclear, but here we show that Aer-2 can interact with the chemotaxis system of Escherichia coli to mediate repellent responses to oxygen, carbon monoxide and nitric oxide. Using this model system to investigate signalling and poly-HAMP function, we determined that the Aer-2 PAS domain binds penta-co-ordinated b-type haem and that reversible signalling requires four of the five HAMP domains. Deleting HAMP 2 and/or 3 resulted in a kinase-off phenotype, whereas deleting HAMP 4 and/or 5 resulted in a kinase-on phenotype. Overall, these data support a model in which ligand-bound Aer-2 PAS and HAMP 2 and 3 act together to relieve inhibition of the kinase control module by HAMP 4 and 5, resulting in the kinase-on state of the Aer-2 receptor. (02/2011) (link)
• Campbell AJ, Watts KJ, Johnson MJ, and Taylor, BL. 2011. Role of the F1 region in the Escherichia coli aerotaxis receptor, Aer. J Bacteriol. 193(2):358-66.    In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor theAer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer. (01/2011) (link)
• Campbell AJ, Watts KJ, Johnson MJ, and Taylor, BL. 2010. Gain-of-function mutations cluster in distinct regions associated with the signaling pathway in the PAS domain of the aerotaxis receptor Aer. Mol Microbiol. 77(3):575-86.    The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14-119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS-FAD domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the beta-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the 'signal-on' state of the PAS domain, and therefore may be markers for the signal-in and signal-out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the beta-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS beta-scaffold and residue Q248C in the HAMP AS-2 helix. (08/2010) (link)
• Airola MV, Watts KJ, and Crane BR. 2010. Identifying divergent HAMP domains and poly-HAMP chains. J Biol Chem. 285(23):le7. (06/2010) (link)
• Airola MV, Watts KJ, Bilwes AM, and Crane BR. 2010. Structure of concatenated HAMP domains provides a mechanism for signal transduction. Structure. 18(4):436-48.  HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.   (03/2010) (link)
• Watts KJ, Johnson MS, and Taylor BL. 2008. Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer. J Bacteriol. 190(6):2118-27.  Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximalsignaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two alpha-helices separated by a structured loop. The proximal signaling domain consisted of two alpha-helices separated by a short undetermined structure. The Af1503 HAMPdomain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for theAer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helixHAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the beta-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed. (03/2008) (link)
• Watts, K. J., Sommer, K., Fry, S. L., Johnson, M. S., and Taylor, B. L.. "Function of the N-terminal cap of the PAS domain in signaling by the aerotaxis receptor Aer." Journal of Bacteriology 188.6 (2006): 2154-2162. Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers. (03/2006) (link)
• Watts, K. J., Johnson, M. S., and Taylor, B. L.. "Minimal requirements for oxygen sensing by the aerotaxis receptor Aer." Molecular Microbiology 59. (2006): 1317-1326. The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared. (02/2006) (link)
• Watts KJ, Ma Q, Johnson MS, and Taylor BL. 2004. Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer. J Bacteriol. 186:7440-7449. The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 α-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer. (11/2004) (link)
• Yu HS, Saw JH, Hou S, Larsen RW, Watts KJ, Johnson MS, Zimmer MA, Ordal GW, Taylor BL, and Alam M. 2002. Aerotactic responses in bacteria to photoreleased oxygen. FEMS Microbiol Lett. 217(2):237-242. Bacterial aerotaxis is a rapid response towards or away from oxygen. Here we report on the use of computer-assisted motion analysis coupled to flash photolysis of caged oxygen to quantify aerotactic responses in bacteria. The caged compound (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl) cobalt(III)] perchlorate liberates molecular oxygen upon irradiation with near-UV light. A mixture of cells and the caged oxygen compound was placed in a capillary tube and challenged by discrete stimuli of molecular oxygen produced by photolysis. We then recorded the rate of change of direction (rcd) as an estimate of tumble frequency in response to liberated oxygen and measured the signal processing (excitation) times in Bacillus subtilis, Bacillus halodurans and Escherichia coli. This computer-assisted caged oxygen assay gives a unique physiological profile of different aerotaxis transducers in bacteria. (12/2002) (link)

Books and Chapters

• Watts KJ and Johnson MS. 2018. Analyzing protein domain interactions in chemoreceptors by in vivo PEGylation. Methods Mol Biol. 1729:137-145. The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivoconditions, maintaining the intracelluar milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia colicells. In this method, E. coliexpressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a largesulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide crosslinking to interact.    The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivoconditions, maintaining the intracelluar milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia colicells. In this method, E. coliexpressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a largesulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide crosslinking to interact.  (10/2018) (link)
• Taylor BL, Watts KJ, Johnson MS. "Oxygen and redox sensing by two-component systems that regulate behavioral responses: behavioral assays and structural studies of aer using in vivo disulfide cross-linking." Methods in Enzymology 422.Part A (2007): 190-232. A remarkable increase in the number of annotated aerotaxis (oxygen-seeking) and redox taxis sensors can be attributed to recent advances in bacterial genomics. However, in silico predictions should be supported by behavioral assays and genetic analyses that confirm an aerotaxis or redox taxis function. This chapter presents a collection of procedures that have been highly successful in characterizing aerotaxis and redox taxis in Escherichia coli. The methods are described in enough detail to enable investigators of other species to adapt the procedures for their use. A gas flow cell is used to quantitate the temporal responses of bacteria to a step increase or decrease in oxygen partial pressure or redox potential. Bacterial behavior in spatial gradients is analyzed using optically flat capillaries and soft agar plates (succinate agar or tryptone agar). We describe two approaches to estimate the preferred partial pressure of oxygen that attracts a bacterial species; this concentration is important for understanding microbial ecology. At the molecular level, we describe procedures used to determine the structure and topology of Aer, a membrane receptor for aerotaxis. Cysteine-scanning mutagenesis and in vivo disulfide cross-linking procedures utilize the oxidant Cu(II)-(1,10-phenanthroline)3 and bifunctional sulfhydryl-reactive probes. Finally, we describe methods used to determine the boundaries of transmembrane segments of receptors such as Aer. These include 5-iodoacetamidofluorescein, 4-acetamido-4-disulfonic acid, disodium salt (AMS), and methoxy polyethylene glycol maleimide, a 5-kDa molecular mass probe that alters the mobility of Aer on SDS-PAGE. (01/2007) (link)
• Taylor BL, Johnson MS, and Watts KJ. 2003. Signal transduction in Prokaryotic PAS Domains, pp 17-50 In ST. Crews (ed.), PAS Proteins: Regulators and Sensors of Development and Physiology. Kluwer Academic Publishers, Norwell, MA. (12/2003)