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Inverted Signaling by Bacterial Chemotaxis Receptors

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Inverted Signaling by Bacterial Chemotaxis Receptors ARTICLE DOI: 10.1038/s41467-018-05335-w OPEN Inverted signaling by bacterial chemotaxis receptors Shuangyu Bi1, Fan Jin1 & Victor Sourjik1 Microorganisms use transmembrane sensory receptors to perceive a wide range of envir- onmental factors. It is unclear how rapidly the sensory properties of these receptors can be modified when microorganisms adapt to novel environments. Here, we demonstrate Escherichia coli 1234567890():,; experimentally that the response of an chemotaxis receptor to its chemical ligands can be easily inverted by mutations at several sites along receptor sequence. We also perform molecular dynamics simulations to shed light on the mechanism of the trans- membrane signaling by E. coli chemoreceptors. Finally, we use receptors with inverted signaling to map determinants that enable the same receptor to sense multiple environmental factors, including metal ions, aromatic compounds, osmotic pressure, and salt ions. Our findings demonstrate high plasticity of signaling and provide further insights into the mechanisms of stimulus sensing and processing by bacterial chemoreceptors. 1 Max Planck Institute for Terrestrial Microbiology & LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg 35043, Germany. These authors contributed equally: Shuangyu Bi, Fan Jin. Correspondence and requests for materials should be addressed to V.S. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:2927 | DOI: 10.1038/s41467-018-05335-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05335-w ransmembrane receptors are ubiquitously used by pro- dephosphorylates CheY and an adaptation system that consists of Tkaryotes to monitor their environment. Elucidating how the methyltransferase CheR and the methylesterase CheB, which receptors sense environmental stimuli is thus essential for modify receptors on several specific glutamyl residues. understanding bacterial adaptation to various environmental Chemotaxis receptors have a modular structure (Fig. 1a) that is niches. Important models for investigation of sensory mechan- typically conserved throughout bacteria and archaea3. Variation isms are chemotaxis receptors, which control cell motility to in the sequence and structure of periplasmic sensory domains enable prokaryotes to accumulate towards favorable conditions1,2. enables chemoreceptors to accommodate many different types of This control is exerted by regulation of the autophosphorylation ligands3,4.InE. coli chemoreceptors, sensory domains are dimers of the receptor-associated kinase CheA, which subsequently of pseudo-four-helix bundles (α1–α4 and α1′–α4′)5, whereby α1/ donates the phosphoryl group to the CheY response regulator α1′ and α4/α4′ extend into membrane as transmembrane helices that controls rotation of flagellar motor. In Escherichia coli, the TM1/TM1′ and TM2/TM2′, respectively. Ligand binding has best-studied model for chemotactic signaling, positive chemo- been shown to promote a 1–2 Å inward piston displacement of tactic stimuli (attractants) inhibit the activity of CheA, whereas the α4-TM2 helix relative to the α1-TM1 helix2,6,7. Signals are negative stimuli (repellents) stimulate it. The chemotaxis signal- further transduced by the HAMP homodimer domain, which is a ing pathway in E. coli further includes the phosphatase CheZ that four-helix, parallel coiled coil composed of two amphipathic a b Periplasmic Ligand-binding pocket TM2 domain A 198 L 199 F A Q W Q L A V I V V V L I L L V A W Y TarTM2-2 No Transmembrane V 200 V 201 TM2-1 response helices V F A Q W Q L A V I A V V V L I L L V A W Y Tar L TM2 HAMP domain I L F A Q W Q L A V I A L V V V L I L L V A W Y Wild-type Tar Attractant L V200IV201I response V F A Q W Q L A V I A L I I V L I L L V A W Y Tar A MH bundle Aromatic W 209 TM2+1I anchor Y 210 F A Q W Q L A V I A L I V V V L I L L V A W Y Tar G TM2+2I I Control F A Q W Q L A V I A L IIV V V L I L L V A W Y Tar Flexible bundle R R cable TM2+2A Repellent M F A Q W Q L A V I A L A A V V V L I L L V A W Y Tar Protein contact L response region L AS1 F A Q W Q L A V I A L I IIV V V L I L L V A W Y TarTM2+3I T c Wild-type Tar/MeAsp d TarTM2+2I/MeAsp 2.05 μ μ μ 0.1 μM 0.3 μM 1 μM 3 μM 10 μM 3 m 30 m 300 m 3 mM 1.80 1.0 1.0 2.00 10 μm 100 μm 1 mM 1.75 0.8 1.95 0.8 1.90 0.6 0.6 1.70 1.85 0.4 0.4 YFP/CFP 1.65 YFP/CFP 1.80 0.2 1.75 Initial FRET response 0.2 Initial FRET response 1.60 1.70 0.0 0.0 1.55 1.65 0 200 400 600 800 1000 10–3 10–2 10–1 100 101 102 103 104 105 0 1000 2000 3000 4000 5000 10–3 10–2 10–1 100 101 102 103 104 105 Time (s) MeAsp (μM) Time (s) MeAsp (μM) ef50 TM2+2I Wild-type Tar Tar 1.1 MeAsp 40 MeAsp t = 0 min t = 0 min 1.0 30 0.9 20 0.8 t = 30 min t = 30 min Relative intensity 10 Relative intensity 0.7 0 0.6 X µ 010203040 50 60 100 m X 100 µm 0 10203040 50 60 Time (min) Time (min) Fig. 1 Effects of mutations in TM2 on Tar response to MeAsp. a Schematic representation of E. coli Tar receptor. b Sequence alignment for the wild-type and mutant Tar. Different colors indicate receptors that mediate attractant, repellent, or no responses to MeAsp, respectively. c, d FRET responses of E. coli CheR+CheB+ strain VS181, expressing the wild-type Tar (c) or TarTM2+2I (d) as a sole receptor, as well as the FRET pair, to a stepwise addition (down arrow) and subsequent removal (up arrow) of the indicated concentrations of MeAsp. The corresponding amplitudes of initial FRET responses were plotted as a function of step changes in concentration of MeAsp. The data were normalized to the saturated response. Error bars indicate standard deviation of three independent biological replicates; note that for most data points error bars are smaller than the symbol size. e, f Microfluidic assay of the chemotactic response of receptorless strain UU1250 expressing wild-type Tar (e) and TarTM2+2I (f) to MeAsp. Cell distribution in the observation channel was recorded when the source is 10 mM MeAsp or without MeAsp (scale bar, 100 μm). The x-component (black arrow) indicates the direction up the concentration gradient of MeAsp. Relative cell density (fluorescence intensity) in the observation channel for the wild-type Tar (e) or TarTM2+2I (f) responding to MeAsp (closed squares) or without MeAsp (open squares) were plotted over time. The cell density in the observation channel before MeAsp stimulating (t = 0) was normalized to be one. Error bars indicate standard deviation of three independent biological replicates 2 NATURE COMMUNICATIONS | (2018) 9:2927 | DOI: 10.1038/s41467-018-05335-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05335-w ARTICLE helices AS1/AS1′ and AS2/AS2′8,9. The input and output sig- oxygen52, or cytoplasmic pH32,53, none of these mutations naling of the HAMP domain plays a central role in transmission inverted the canonical ligand response, and the interpretation of of signals10,11. A junction between TM2 and AS1 of the HAMP the observed response inversion remained unclear. domain is formed by a short control cable that extends from TM2 In contrast to these previous studies, here we demonstrate that and has helical character12,13. Changes in the helicity of this sequence changes in the TM2 helix and in the region after the control cable were proposed to be important in transmitting the HAMP domain of Tar can easily invert its response to canonical piston motion of TM2 and modulating the conformation of the amino acid ligands. Based on molecular dynamics (MD) simu- HAMP domain12–16. Subsequently, signals are transduced lations, we propose mechanisms behind this striking plasticity of through the methylation helix (MH) bundle, the flexible region, bacterial receptors, thereby providing further insights into signal and towards the protein contact region interacting with CheA transduction between receptor domains. Finally, we demonstrate and the adaptor protein CheW, with all of these regions organized how Tar mutants with inverted signaling can be used as tools to as one anti-parallel four-helix bundle17,18. Structural dynamics of identify receptor determinants that are responsible for detecting these cytoplasmic regions are thought to be important in mod- various types of environmental stimuli. ulating the kinase activity19–25. In addition to ligand binding, the activity of chemoreceptors also depends on the level of receptor Results methylation that is controlled by the adaptation system. CheR Extension of TM2 inverts the response of Tar. To better preferentially methylates receptors that have inactive (kinase- understand the mechanism of transmembrane signaling in E. coli inhibitory) conformation and CheB demethylates active recep- chemoreceptors, we constructed a series of Tar mutants where the tors, respectively, increasing or decreasing their activity. These TM2 helix was shortened by one or two amino acids or extended negative feedbacks enable the chemotaxis pathway to tune by up to three amino acids (Fig.
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